ENVIRONMENTAL TOXICOLOGY

STUDY GUIDE for BIOCHEMISTRY

2009

UNIVERSITI TEKNOLOGI MARA

FACULTY OF HEALTH SCIENCES

BIOCHEMISTRY FOR ENVIRONMENTAL HEALTH

INSTITUT PERKEMBANGAN PENDIDIKAN (InED)

UNIVERSITI TEKNOLOGI MARA (UiTM)

40450 SHAH ALAM

BIOCHEMISTRY FOR ENVIRONMENTAL HEALTH (ENV 400/416)

Bachelor in Environmental Safety and Health (Honours) Program (e- pjj)

Faculty of Health Sciences

Universiti Teknologi Mara (UiTM)

Course Description:

This is an introduction to the chemistry of biological compounds. A systematic study of carbohydrates, lipids, amino-acids, proteins, nucleic acids, and their components is presented. Metabolism of the biological compounds is also studied as are the interrelations among the carbon, nitrogen, and energy cycles. Enzymology, intermediary metabolism, and metabolic control will also be included.

Course Outcomes:

Upon successful completion of this course the student should be able to:

1.Explain the different types of bonding and interactions found in biochemistry such as hydrogen bonds, ionic bonds, hydrophobic interaction, Van der Waals forces and asymmetry of carbon compounds with cis-trans isomerism.

2.Explain that water molecules are polar and form irregular hydrogen-bonded networks in liquid state and why polar and ionic substances dissolve in water.

3.Explain how acids and bases affect the pH of a solution, the relationship between pH and pK for a solution of weak acid and how the buffer works.

4.Describe the structure of an amino acid and the structure of the 20 different R groups.

5.Describe the structure of alpha helix, beta sheet primary, secondary, tertiary and quartenary and the covalent and non-covalent forces that maintain structures.

6.Describe the metabolic disorder, phenyketonuria.

7.Explain how the Michaelis-Menten equation relates the initial velocity of a reaction for an enzyme substrate reaction and Lineweaver plot can be used to present kinetic data.

8.Describe competitive and non-competitive inhibitor.

9.Explain the common features in amino acid biosynthesis and the role of urea cycle in amino acid breakdown.

10.Explain the levels of nucleic acid structure and the structure and functions of DNA and RNA

11..Describe how monosaccharide cyclize to from two different anomers and the monosaccharide linkages in polysaccharides.

12.Describe lactose intolerance, diabetes and hypoglycemia

13.Describe glycolysis and the citric acid cycle to synthesize ATP and some allied health perspective of anerobic metabolism with dental plaque.

14.Describe electron carriers and how electrons travel from the different complexes.

15.Describe the connection between Electron Transport Chain and Oxidative Phosphorylation.

16.Describe the structure and nomenclature of lipids including fatty acids, triacyglycerols, sphingolipids and phophoglycerides.

17.Explain the physiological roles of lipids as membrane components and energy storage molecules.

18.Explain lipid of lung surfactant.

19.Explain fatty acid synthesis and degradation.

20.Explain cholesterol biosynthesis and atherosclerosis

.

CONTENTS

PAGE

1.0 Basic Aspects of the chemistry of life

1.1 Biochemistry as the chemistry of living systems

1.2 Asymmetry of carbon compounds and cis-trans isomerism

1.3 Different types of bonding such as hydrogen bonds, ionic bonds hydrophobic interactions, Van der Waals forces

2.0 Water, acid and base, buffer

2.1 Physical properties of water

2.2 Biological importance of water as a solvent

2.3 Hydrogen ion concentration and pH of biological systems

2.4 Relationship between pH and pK for a solution of weak acid

2.5 Physiological buffer systems

3.0 Amino acids and Proteins

3.1 Overall structure and properties of the 20 different R groups.

3.2 Ionizable groups in amino acids.

3.3 Peptide bonds link amino acid residues in a polypeptide

3.4 The structure of primary, secondary, tertiary and quartenary proteins and the covalent and non-covalent forces that maintain structures.

3.5 The metabolic disorder, phenyketonuria

4.0 Properties of Enzymes

4.1 Classification and general catalytic properties of enzymes.

4.2 Michaelis-Menten equation relates to the initial velocity of a reaction for an enzyme substrate reaction

4.3 Lineweaver plot to present kinetic data.

4.4 Competitive and non-competitive inhibitor and examples.

5.0 Nitrogen metabolism

5.1 Common features in amino acid metabolism

5.2 Glucogenic and Ketogenic amino acids

5.3 The role of urea cycle in amino acid breakdown

6.0 Sugar and carbohydrate structure

6.1 Monosaccharide and their derivates.

6.2 Cyclization to from two different anomers and glycosidic bond that links two monossacrides.

6.3 Dissacharides and other sugars example as sweeteners

6.4 Polisaccharides such as starches and glycogen, cellulose.

6.5 Lactose intolerance, diabetes , hyphoglyceamia and hyperglycemia.

7.0 Metabolic processes central to ATP synthesis- Glycolysis and Citric acid cycle

7.1 Glycolysis involves the breakdown of glucose to pyruvate to synthesize ATP

7.2 Aerobic and anaerobic metabolism.

7.3 The citric acid cycle, a multistep catalytic process that converts acetyl groups to NADH, FADH and GTP.

7.4 Allied health perspective of anaerobic metabolism

8.0 Electron transport and oxidative phosphorylation

8.1 Electron carriers as electrons travel from the different complexes.

8.2 The reactions catalyzed by the complexes and their mechanism

8.3 The connection between electron transport chain and oxidative phosphorylation.

9.0 Lipid and Membranes

9.1 Structure and nomenclature of lipids including fatty acids, triacyglycerols, sphingolipids and phophoglycerides

9.2 the physiological roles of lipids as membrane components and energy storage molecules.

9.3 Lipid of lung surfactant

10.0 Lipid metabolism

10.1 Steps of fatty acid synthesis and its mode.

10.2 HMG-CoA is important in cholesterol biosynthesis.

10.3 Atherosclerosis

11.0 Nucleotides, nucleic acids

11.1 Levels of nucleic acid structure nitrogenous bases, nucleosides, nucleotides.

11.2 Structure and functions of DNA and RNA .

11.3 Use of nucleoside analogues as drugs

Syllabus contents

Chapter 1: Basic aspects of the chemistry of life

1.1biochemistry is the chemistry of living systems:

1. complicated and highly organized

2. each part has a function

3. function is related to structure

4. must extract energy from the environment

chemicals of living systems

alcohols

esters

ethers

amides

acids

anhydrides

also include thiols and phosphates

Biochemistry deals with the structure and function of biomolecules

biochemists study the structures of bio-molecules and their cellular functions to better understand living systems and their chemistry

Example of structure-function relationship

1. amino acids are joined to form proteins and these proteins fold up to form functional enzymes

2. nucleotides are joined to form Rna and Dna. these polymers are the information molecules of living systems and maintain the genetic heritage of organisms

3. proteins (enzymes), RNA and DNA along with other molecules aggregate to form cellular components, cells, organs and whole organisms.

Rna comes in 3 basic forms:

tRNA (transfer rna) = adapter in protein synthesis - matches codon to amino acid

Rrna (ribosomal RNA) = structural RNA in ribosomes

mRNA (messenger rna) = contains information for protein synthesis

cell structure

basics of the relationship between proteins and DNA:

linear relationship between DNA, RNA and protein sequence

DNA encodes amino acids of a protein using 3 letter codons.

DNA is transcribed to make mRNA.mRNA is translated by ribosomes to make the protein.

Biochemistry can be divided into 3 areas of study

conformational- structure and 3d arrangements of biomolecules

metabolism energy production and utilization

informational- language for communication inside and between cells

Practical applications of biochemistry

1. in medicine and health care :

enzymes as markers for disease eg lactate dehydrogenase (heart attack can diagnosed by an increase of ldh from heart muscle

acetylcholinesterase (ace) important in controlling certain nerve impulse. many pesticides interfere with this enzyme.

designer drugs new and improved antibiotics and chemotherapy agents

human proteins through genetic recombinant techniques eg insulin, hgh

2. in agriculture herbicides & pesticides, genetic engineering

3. chemical industries synthesis & detoxification

Biochemical connections

lactic acid and sports

neurophysiology some aa are key precursors to hormones and neurotransmitters

nutrition- aspartame (sweetener), lactose intolerance

allied health- phenylketonuria, multiple sclerosis, lupus (autoimmune disease /immune system attacks the body own tissues involve rna processing), dental plaque,anemia, atherosclerosis

forensic- uses of DNA testing

Chapter 2: Water, acid and base, buffer

Water

essential for life

major constituent of almost all life forms

most animals and plants contains more than 60% water by volume

structure consists of 2H atoms bonded to 1 O atom.

the H side of the molecule has a slight +ve and the O side a ve charge exist

makes it polar and has strong solvent properties

hydrophilic compounds interact (disslove) with water eg . polar cpds (alcohols and ketones)& ionic cpds (kcl), amino acids

hydrophobic compounds do not interact with water eg. non polar cpds (hexane, fatty acids, cholesterol)

Roles of water in the life of organisms

mammalian cells 70% water

solvent for biological systems & for most chemical reactions that support life.

75% of the earth is covered with water

has a very high specific heat-retains heat better than other materials

Some uses of water as solvent

flavoring and co2 gas dissolved in water to make soft drinks

farmers use water to dissolve fertilizers

medicines in water

chlorines or flourides added to water

Hydrogen bonds

water molecules are hydrogen bonded

the ability to form strong h bond is responsible for the many unique characteristics of water such as its high melting point and boilng point

3d structures of many important biomolecules including proteins (Hb) and nucleic acids (DNA) are stabilized by H bonds

Acids, bases, and buffers

Principle of ionization of weak acids:

the fundamental concept of buffers is: a buffer resists change

pH buffers resist change in ph when either acid (h+) or base (oh-) is added to it.

chemicals which are ph buffers are weak acids or bases

acids = proton (H+) donors

bases = proton acceptors

This tendency to ionize can be put in terms of an equation for the equilibrium:

where [ ] = molar concentration; k = ionization constant (acid dissociation constant)

Simplest example is water (H2O):

but since [H2O] (water concentration) = constant (55.5 m), kw = [h+][oh-] = 10-14 M

in pure water, [h+] = [oh-] = 10-7 m

to make this easier to use, the ph scale was invented.

pH = -log [h+]; thus when [h+] = 10-7 m, ph = 7

this is called neutral ph because it is in the middle of the ph scale. at ph greater than neutral, the solution is alkaline; while at ph less than neutral, the solution is acid.

Titration of a Weak Acid illustrating its Ionization and Buffering Property

all weak acids have titration curves like this one. bases (like ammonium, nh4+) are also weak acids and have similar titration curves.

the position where the buffering zone is on the ph scale is related to the chemical nature of the weak acid:

acetic acid ionizes in the acidic portion of the pH scale

this relationship is known as the Henderson-Hasselbalch equation.

useful in predicting the properties of buffer solutions used to control the pH of reaction mixtures.

the pk of a weak acid is the ph where [a-] = [ha]

at pH below the pk, [HA] > [A-]

at pH above the pk, [HA] < [A-]

therefore the pk determines the buffering zone for a weak acid.

a similar expression pk can be used, pk=-log k

the ph of a solution of a weak acid and its conjugate base is related to the concentration of the acid and base- Henderson- Hasselbach equation.

for example, acetic acid has a pk = 4.8 and a buffering zone from ph 3.8 to 5.8.

so a weak acid will be an effective buffer at ph = pk +/- 1 ph unit.

summary

acids are proton donors and base are proton acceptors

water can accept or donate protons

the strength of an acid is measured by its acid dissociation constant, k

the larger the k value, the stronger the acid and more h+ dissociates

the conc. h+ is expressed as ph, -ve log of H ion conc.

Calculating pH for weak acids and bases

Calculate the relative amounts of acetic acid and acetate ion present at the following points when 1 mol of acetic acid is titrated with NaoH. use HH eqn. to calculate ph

1. 0.1 mol NaOH added

2. 0.3 mol NaOH added

3. 0.5 mol NaOH added

ratio 1:1, when 0. 1 mol of naoh added, 0.1 mol acetic acid reacts with it to form 0.1 mol acetate ion, leaving 0.9 mol acetic acid

ph = pk + log 0.1/0.9

= 4.76 -0.95

= 3.81

TUTORIAL 1, ENV 416/400

1. Calculate the hydrogen ion concentration for each of the following materials:

a) Blood plasma, pH 7.4

b) Orange juice, pH 3.5

c) Human urine, pH 6.2

d) Household ammonia, ph 11.5

e) Gastric juice, pH 1.8

2. Define the following :

a) Acid dissociation constant

b) Equivalence point

c) Hydrophilic

d) Hydrophobic

e) Non polar

f) Polar

3.What is the [CH3COO-] / CH3COOH ratio in an acetate buffer at pH 5.00?

4. What are some macromolecules that have hydrogen bonds as part of their structures?

5. What is the relationship between pKa and the useful range of a buffer?

6 What is the pK of a weak acid HA if a solution containing 0.2M HA and 0.1M A- has a pH 0f 6.5 ?

7. Explain buffer solution. Give an example of a buffer solution.

8. Explain why polar substances dissolve in water while non polar substances do not.

9. Explain why a 1M solution of HCl has a pH of 0.

10. A 5.0 ml of H2SO4 is titrated with 0.2 M KOH to neutrality. If 4.5 ml of KOH was used what was the pH of the original acid?

Chapter 3: Amino acids and Proteins

Amino acids and peptides1. Only 20 aa usually found in proteins2. The general structure includes an amino group and a carboxyl gp.3. The -carbon is bonded to a H and side chain gp (R)4. The R gp determines the identity of the particular amino acid

Types of Amino Acids based on side-chain chemical character:

I. Non-Polar or hydrophobic (water hating)

II. Flexible

III. Polar or hydrophilic (water loving)

There are 20 Amino Acids encoded by codons in the genetic code:

When there are more than 100 AAs found in nature, why only 20 AAs in proteins? Because these 20 AAs provide all the chemical and size groups needed to make a very large number of proteins. Plus many of these amino acids become modified after translation into proteins, which increases the available chemical character of amino acid side chains.

These 20 AAs can be divided into the above 3 groups (non-polar, flexible and polar) and then subdivided by their chemical character:

Group I = Non-Polar -- 8 AAs

Hydrocarbon NON-POLAR AMINO ACIDS -- 5 AAs -- Ala Val Leu Ile Pro:

Non-Polar -- Hydrocarbon -- Ala (Alanine)

The chiral Carbon of Ala is emphasized here! All amino acids are derivatives of Ala, except Gly

Non-Polar -- Hydrocarbon -- Val (Valine)

Val has to methyl groups added to Ala to make an isopropyl group.

Non-Polar -- Hydrocarbon -- Leu (Leucine)

Leu adds an isopropyl group to Ala so that Leu has 4 carbons in its side chain.

Non-Polar -- Hydrocarbon -- Ile (Isoleucine)

Ile is a structural isomer of Leu so it also has 4 carbons in its side chain. But Ile is bulkier than Leu near the base of the side chain, while Leu is bulkier than Ile farther out on the side chain (size/shape of side chains is important). Ile has a 2nd chiral center which is emphasized in the Ile drawing above.

Non-Polar -- Hydrocarbon -- Pro (Proline)

Pro is a very special amino acid due to its inflexible character!!! Pro is inflexible because its side chain bonds to alpha-amino group in a ring structure which can not twist around the bond between alpha-amino group and alpha carbon, which all other AAs can. Also Pro, thus, has a secondary amino group (notice the single hydrogen on its Nitrogen atom) with different chemical character than the primary amino groups in all other amino acids, which have two hydrogens on them.

Aromatic NON-POLAR AMINO ACIDS -- 2 AAs --Phe

Trp:

Non-Polar -- Aromatic -- Phe (Phenylalanine)

Phe adds a benzene ring to Ala!

Non-Polar -- Aromatic -- Trp (Tryptophan)

Trp has a heterocyclic aromatic group with an aromatic amine in it.

Thiol Ether NON-POLAR AMINO ACID -- 1 AA -- Met:

Non-Polar -- Thiol Ether -- Met (Methionine)

Met introduces the important Sulfur element into proteins which is found in Cys also (see

below). Met contains a thiol ether (R-S-R) in its side chain, which is much less polar than an oxy-ether (R-O-R) like the compound we call ether, which is an polar organic solvent. Met is a very hydrophobic AA.

Group II = Flexible -- 1 AA -- Glycine is the Flexible Amino

Acid

Flexible -- Gly (Glycine)

Gly is a unique AA with no chiral center -- but it is prochiral since it has two groups the same (ie H) on the central carbon -- so it still has sidedness - try making a model of Gly. Most important since Gly has no side chain it is very flexible and can easily twist around its alpha-amino Nitrogen bond to the alpha-Carbon. Gly is the opposite of Pro - Gly is flexible while Pro is inflexible.

Finally, Gly makes a transition from the non-polar AAs to the polar AAs. Gly is neither nonpolar or polar .

Group III = Polar -- 11 AAs

THE POLAR AMINO ACIDS

Polar AAs are important since they provide chemical groups for interaction with water. Thus, the hydrogen bonding character of polar AAs is key in forming protein structures. While the ionic bonding character of charged polar AAs is also important in protein structure. Also the polar side chains in these AAs provide the chemically reactive groups in proteins.

Alcohols - Neutral Polar Amino Acids -- 3 AAs -- Ser Thr & Tyr:

Polar -- Neutral -- Alcohols -- Ser (Serine)

Ser contains one -OH group and so it is essentially hydroxy-Ala. The hydroxyl group on Ser does not normally ionize, so it is not charged in proteins - its neutral. Ser is the smallest AA of the polar amino acids and is very polar. The hydroxyl group on Ser provides enzymes a very good nucleophilic group for doing chemistry. Another important function of Ser is to form esters with phosphate, making phospho-ester proteins. Phosphorylation of proteins/enzymes is very important in regulation of activity.

Polar -- Neutral -- Alcohols -- Thr (Threonine)

Thr adds a Carbon on to Ser, which makes the hydroxyl group less accessible in Thr than Ser. Thr serves more often in a structural role in proteins and is usually not as chemically active as Ser. Thr can form esters with phosphoric acid and phospho-Thr is often found in proteins.

Polar -- Neutral -- Alcohols -- Tyr (Tyrosine)

Tyr is an aromatic alcohol and so it has both aromatic character and polar character. The

hydroxyl of Tyr is like the hydroxyl in phenol, so at high pH it can ionize. Tyr can also form phospho-esters like Ser and Thr. Phospho-Tyr is very important in proteins/enzymes involved in regulating the cycle cell.

Thiol - Neutral Polar Amino Acid -- 1 AA -- Cys:

Polar -- Neutral -- Thiol -- Cys (Cysteine)

Cys is essentially thiol-Ala. The thiol (-SH) group of Cys can ionize as shown in graphic. Thiols ionize at about pH 8 and so usually they are protonated at biological pH. Hydroxyl groups like in Ser have pK about 15 or so and do not ionize normally.

A Special Feature of Cys is that it can oxidize (in the presence of oxygen) and

react with another Cys to form Cystine or a disulfide bond:

The formation of "Cystine" can take place between 2 polypeptide chains to make a cross-link between them. This is actually an enzyme catalyzed reaction which takes place in the lumen of ER in cells when proteins are being exported from the cell. A very good example is the production of antibodies by cells in the immune response - antibody proteins contain many Cys- Cys or disulfide bonds. Excellular proteins often contain Cys-Cys bonds, while cellular proteins do not usually contain the Cys-Cys since the conditions in the cell are reducing. In the second part of the graphic above, the general reaction of 2 thiols is shown. In the presence of oxygen or oxidizing conditions, the 2 thiols react to form a disulfide bond between them. Since this is a redox reaction, the hydride ion released by each thiol is usually coupled to an electron acceptor reaction or in simple oxidiation with oxygen, hydrogen peroxide is usually formed with further reduction to water.

Amides - Neutral Polar Amino Acids -- 2 AAs -- Asn & Gln:

Polar -- Neutral -- Amides -- Asn (Asparagine)

Asn is a very small amino acid as well as being very polar. Amides are neutral and do not ionize nor do they accept protons.

Polar -- Neutral -- Amides -- Gln (Glutamine)

Gln is a bit larger amide than Asn because it has a longer side chain string of Carbons. Both the amide AAs are neutral derivatives of the corresponding acid AAs (Asp & Glu - see below) Understanding the chemical character of the amide is very important, since the peptide bond of proteins is an amide bond.

Acids - Negatively Charged Amino Acids -- 2 AAs -- Asp & Glu:

Polar -- Charged -- Acids -- Asp (Aspartic acid or Aspartate)

Asp has a second carboxylic acid group in addition to its alpha-carboxylic acid group. The Asp side chain carboxyl group is normally ionized at biological pH; Asp a negatively charged AA. Asp is a rather small AA and is very polar.

Polar -- Charged -- Acids -- Glu (Glutamic acid or glutamate)

Glu also has a second carboxylic acid group in addition to its alpha-carboxylic acid group. The Glu side chain carboxyl group is normally ionized at biological pH; Glu is negatively charged.

Bases - Positively Charged Amino Acids -- 3 AAs -- Lys Arg & His:

Polar -- Charged -- Bases -- Lys (Lysine)

Lys has a primary amino group at the end of a 4 Carbon side chain and it can be positively

charged. Since the Lys side chain amino group has a high pK , it is often charged

at biological pH.

Polar -- Charged -- Bases -- Arg (Arginine)

Arg has a complex side chain containing 3 Nitrogen groups, which work as a unit to give a positive charge. Since the Arg side chain group has a very high pK , it is always

charged at biological pH. Arg provides proteins/enzymes with essentially a fixed positive charge.

Polar -- Charged -- Bases -- His (Histidine)

His has an aromatic-like pair of amino groups, making His a unique AA with a positive charge -- sometimes. His with a pK for its side chain near neutrality, means that it can either be charged or not at biological pH. His, when not charged, is a very strong nucleophile and is very important in enzyme chemistry. His is also very important as a proton acceptor and donor in biochemical

reactions.

Protein Covalent Structure (Protein Primary Structure)

I. Peptide Bonds, Peptides and Proteins

Proteins are sometimes called Polypeptides, since they contain many Peptide Bonds

The peptide bond is an amide bond

Water is lost in forming an amide bond.

Structural Character of Amide Groups: Understanding the chemical character of the

amide is very important, since the peptide bond of proteins is an amide bond.

Amides have a partial double bond character and also a partial charge character because of the resonance forms shown in the above graphic.

Comparison of an amino acid, a dipeptide and a tripeptide:

Amino Acid = Gly; dipeptide = Gly-Ala; tripeptide = Gly-Ala-Ser

Peptides = Mini-Proteins

A pentapeptide -- GlyAlaSerPheGln

1st amino acid is always written on the left and called the Amino terminal, since it is always the only amino acid of the peptide with a free alpha-amino group. Last amino acid is always written on the right and called the Carboxyl terminus, since it is always the only amino acid of the peptide with a free alpha-carboxylic acid group.

List of Proteins Shown in Amino Acid Composition Table:

A. Antibody - Human Bence-Jones Kappa (antibody light chain)

B. Human Cytochrome c (electron transport protein)

C. Spinach Ferredoxin (electron transport protein)

D. Pig Glucagon (protein hormone)

E. Bovine Insulin (protein hormone)

F. Human/Gorilla Hemoglobin alpha chain (oxygen transport protein)

G. Human/Gorilla Hemoglobin beta chain (oxygen transport protein)

H. Chicken Lysozyme (enzyme)

I. Sheep Wool (structural protein)

Free amino acids are obtained from proteins by strong acid hydrolysis:

B. OVERALL CONFORMATION OF PROTEINS

Proteins have a covalently bonded backbone as discussed in Lecture 5 in relation to amino acid sequence determination. But the 3-D shape or conformation is held together by weaker bonding of the non-covalent type. The linear form of the polypeptide backbone of the protein folds into a tightly held shape which is chemically stabilized by weak bonds like hydrogen bonds, ionic bonds and hydrophobic interactions among non-polar amino acid side chains.

To reduce the complexity of protein structure to a manageable level for our study and

understanding, the protein is considered to have 4 levels of structure.

Four Levels of Protein Structure:

1. Primary Structure- Polypeptide backbone

2. Secondary Structure- Local Hydrogen bonds along the backbone

3. Tertiary structure- Long distance bonding involving the AA side chains

4. Quaternary structure- Protein-Protein interactions leading to formation of dimers,

tetramers, etc.

C. PRIMARY STRUCTURE OF PROTEINS

We have already discussed the Primary structure of Proteins, which is the polypeptide backbone or amino acid sequence. The amide bonds joining the individual amino acid residues of the backbone have an important role in forming the 3-D structure of proteins. The peptide bond of the amino acid sequence forms a planar structure due to the partial double bond between N and C. This planar structure limits the ways the backbone can fold up and therefore, constrains the shape a folded polypeptide can take.

The Amide Bond showing its partial double bond character and partial charges.

D. SECONDARY STRUCTURE OF PROTEINS

In 1950's, Linus Pauling named the first structures he found by X-ray diffraction, the Alpha Helix and the second structure he found was called Beta Sheet. We continue to use these names today for two forms of secondary structure and add a third type forms in regions where the protein bends back on itself to form its compact shape or conformation.

The 3 Types of Protein Secondary Structure:

Alpha-helix

Beta-sheet

Turns or Bends (Bends in backbone to fold the polypeptide back on itself)

E. LOCAL HYDROGEN BONDING FORMS SECONDARY STRUCTURE

Secondary Structure is formed by local Hydrogen Bonding between the Hydrogen on the

Nitrogen of one amide in a peptide bond with carbonyl oxygen of another amide in a second peptide bond.

Hydrogen bonds (H-bonds)are weak non-covalent bonds. The energy required to break an Hbond is about 1 to 4 kcal/mole as compared to a covalent bond which requires about 100

kcal/mole to break. Thus, H-bonds are a bit flexible and for example, the H-bonds holding water together as liquid constantly break and reform. However, in more directed H-bonds like found in protein secondary structure, the pair of groups involved stay as partners and with the overall arrangement of a single H-bond being stabilized by a group of H-bonds. So H-bonding in secondary structure is stronger due to the local grouping of these bonds and secondary structure forms like the alpha-helix and beta-sheet are neighborhoods of H-bonds acting together like a group.

Figure 7. Hydrogen Bond (H-Bond) between Two Peptide Bonds.

.

Model of Right-Handed Alpha-Helix Showing H-Bonding (From Voet/Biochemistry

1990 John Wiley)

Model of Beta Sheet Showing H-Bonding between Two Strands of the Sheet. (From

Voet/Biochemistry 1990 John Wiley)

F. Alpha-HELIX

Alpha helix is held together by hydrogen bonds between the amide Hydrogen on the Nitrogen and another amide carbonyl oxygen of every 4th amino acid residue (approximately). These are intrachain H-bonds which along the same region of the backbone of the polypeptide or in other words within the same region of the amino acid sequence.

Simple Model of Alpha Helix with H-bonding Pattern.

The side chains of the amino acids project out from the core of the alpha helix. Water is excluded from the tight inner core of the alpha helix, which is very hydrophobic.

G. Beta SHEET

Beta sheets are also held together by hydrogen bonds between the Hydrogen on the Nitrogen and another amide carbonyl oxygen of the peptide bonds but between chains of the backbone rather than along it as was found for the Alpha helix. These are called interchain H-bonds since they form between two parts of the polypeptide backbone separated from one another by some distance or length of the amino acid sequence of the polypeptide.

Simple model of H-Bonding in a Beta Sheet.

Two types of backbone chain order is found:

1. PARALLEL where the chains run in the same direction

2. ANTI-PARALLEL where chains run in the opposite direction

Models of (a) Antiparallel and (b) Parallel Beta Sheets (Only two strands of beta-sheet

shown).(From Voet/Biochemistry 1990 John Wiley)

H. TURNS AND BENDS IN THE POLYPEPTIDE BACKBONE

Proline (Pro) breaks up secondary structures like alpha-helix and beta-sheet. Because Pro can not bend, Pro is often found at the ends of Alpha Helix and Beta Sheet strands. Thus, the third type of Secondary Structure is actually formed by the absence of the other two types.

Positions of Pro in Relation to Alpha-Helix and Beta Sheet Secondary Structures

Places where the polypeptide backbone bends so that the protein can fold back on itself to form the compact structure also have hydrogen bonds in some cases. These H-bonds occur only between the 1st and 4th amino acid residue of the Reverse Turn and no other H-bonds are formed.

TUTORIAL 2, ENV 416

1. Draw the dipeptide Asp-His

2. Identify the nonpolar amino acids and the acidic amino acids in the following peptide :

Glu-Thr-Val-Asp-Ile-Ser-Ala

3. Sketch a titration curve for alanine and indicate the pKa values for all the titratable groups. Also indicate the pH at which this amino acid has no net charge.

4. Draw 2 hydrogen bonds, one is part of a secondary structure and another that is part of a tertiary structure.

5. Draw a disulfide bridge between two cysteines in a polypeptide chain.

6. What is the highest level of oragnization in myoglobin and hemoglobin?

7. Differentiate between secondary and tertiary proteins. Name an example for each.

8. Differentiate between alpha-helix and beta sheet.

CHAPTER 4: PROPERTIES OF ENZYMES

Enzymes are biological catalysts. Like all catalysts, enzymes lower the energy needed to get a reaction started. Enzymes are much generally better at accelerating the rates of reactions than non-biological catalysts.

Figure 1. Diagram showing that less energy is required to get an enzyme catalyzed reaction started as compared to a non-catalyzed reaction. Figure from Zubay et al., Principles of Biochemsitry copyright 1995 Brown Comm.

Enzymes have been divided into 6 classes by the International Commission on Enzyme

Nomenclature. All enzymes are assigned a number (called an EC number) which defines exactly the reaction catalyzed by the enzyme. For example, trypsin is EC 3.4.21.4 since it is in class 3 (hydrolases) which work on peptide bonds (3.4) in the middle of proteins (3.4.21 are serine endopeptidases) - trypsin is the 4th entry in this subclass.

These six classes are:

1. Oxidoreductases - enzymes catalyzing oxidation reduction reactions.

2. Transferases - enzymes catalyzing transfer of functional groups.

3. Hydrolases - enzymes catalyzing hydrolysis reactions.

4. Lyases - enzymes catalyzing group elimination reactions to form double bonds.

5. Isomerases - enzymes catalyzing isomerizations (bond rearrangements).

6. Ligases - enzymes catalyzing bond formation reactions couples with ATP hydrolysis.

These 6 enzyme classes can also be illustrated by the general reactions catalyzed:

Figure 2. Model reactions of the 6 classes of enzymes. Figure from Zubay et al., Principles of Biochemsitry copyright 1995 Brown Comm.

Examples of enzymes in each class:

1. Alcohol dehydrogenase (EC 1.1.1.1)

2. Hexokinase (EC 2.7.1.1)

3. Trypsin (EC 3.4.21.4)

4. Ribulose-bisphosphate carboxylase (EC 4.1.1.39)

5. Triose phosphate isomerase (EC 5.3.1.1)

6. Tyrosine tRNA ligase (6.1.1.1)

Enzyme Additives (Cofactors) Assisting in Catalysis

Enzymes are often composed of only protein. In this case only AA side chains are used for catalysis. Some enzymes require additives for assisting with catalysis. Additives like vitamins often provide functional groups not available to the enzyme among the side chains of the amino acids.

In these cases the protein of the enzyme binds:

Organic cofactors (Vitamins = organic cofactors)

Metal ions (e.g. Mg2+)

Nucleotides (even RNA)

The Common Cofactors (Enzyme Additives):

Biotin aids in carboxylation reactions (carbon dioxide fixation).

Cobaltamine (vitamin B-12) aids in alkylation reactions (methylation for instance).

Coenzyme A aids in acyl transfers like in the tricarboxylic acid cycle.

Flavin (vitamin B-2) aids in oxidation-reduction reactions (e.g. nitrate reductase).

Lipoic acid aids in acyl transfers via oxidation-reduction processes.

Nicotinamide coenzymes like NAD+ act as independent co-substrates.

Pyridoxal (vitamin B-6) aids in amino group transfers (provides aldehyde functional

group).

Tetrahydrofolate aids in one-carbon transfers.

Thiamin (vitamin B-1) aids in aldehyde transfers and alpha-keto-acids decarboxylations

The complex of protein and additive is called Holo-Enzyme. When the additive is removed from the enzyme, the remaining protein part of the enzyme is called the Apo-Enzyme.

Apo-Enzyme (inactive) + Additive = Holo-Enzyme (active)

The Active Site of the Enzyme.

Each enzyme has a unique active site.

Active site = catalytic site.

The enzyme binds its substrate(s) at the active site and the enzyme catalyzes chemical changes in the substrate(s). The types of chemical reactions catalyzed were illustrated above in

Figure 3. NAD+ bound in the active site of GAP dehydrogenase. The NAD+ molecule is shown in bold and the side chains of the amino acids binding it are shown projecting from the surface of the enzyme (shown as the filled in area surrounding the active site).

Enzymes contain a large number of amino acids, but most AA side chains are used for forming the enzyme's shape. Only a few AA side chains are at the active site. These special AA side chains:

1. Bind the substrate(s) and

2. Catalyze the reaction

This concept is illustrated in the following figures by 3 different drawings of the enzyme

ribonuclease which catalyzes the hydrolysis of RNA. The first view is of the 3-D shape of the enzyme with the 3 key amino acids at the active site highlighted (His12, Lys41 and His119 - numbers indicating the position of these residues in the amino acid sequence of ribonuclease).

Next is a ribbon model with the 3 key amino acids shown in relation to the various secondary structure elements of ribonuclease. Last is a ball-and-stick model of ribonuclease with the same 3 amino acid side chains of the active site emphasized. A feature to try to see in these models is the groove of the enzyme which forms the active site and how the enzyme folds to bring these 3 key amino acid side chains together to form the active site.

Figure 5. 3-D model of the enzyme ribonuclease with the key amino acid side chains at the active site shown in red. The active site is a deep groove at the center of this structure.

Summary of the Active Site of Enzymes:

Enzyme has large structure with hundreds of AA side chains but only a few are involved

in catalysis.

Each enzyme has a unique active site.

Key AA side chains are involved in binding and catalysis in the active site.

Enzyme Framework - Why are Enzymes so Large?

We have discussed the formation of a protein's 3-D shape recall- the 4 levels of protein structure: Primary, Secondary, Tertiary, Quaternary. They make up a "Framework" to bring the AA side chains of the active site together. By bringing the AA side chains of the active site together they can act synergistically or in concert which is part of what makes enzymes very effective catalysts.

Figure 6. Ribonuclease with substrate RNA model bound in active site. His12 and His119 are involved in catalysis of the phosphodiester bond in the backbone of the RNA. Lys41 assists with binding the RNA molecule.

The active AA side chains also provide the enzyme with a high degree of specificity so that only certain substrates are bound to the enzyme's active site..

How do enzymes catalyze a reaction???

One answer is: Like all catalysts, enzymes decrease the energy required to get a reaction started. This was illustrated in the first part of this lecture with an energy diagram. Below is shown a similar diagram with more detail for the energy pattern for the enzyme catalyzed reaction. First, energy is required to form the complex between the enzyme and substrate (E-S complex) which is a higher energy state than the free enzyme and substrate/product.

Figure 7. Diagram of energetics of enzyme catalyzed reaction versus non-catalyzed reaction.

Summary of Enzyme Catalysis:

Enzymes bind substrate with great specificity

Enzyme catalyzed reactions usually have no side products

Enzymes use energy released when substrates bind to make their catalysis more effective.

Introduction to Enzyme Kinetics.

In chemistry, kinetics has to do with the rate of reactions. In biochemistry, we are most interested in rates of enzyme catalyzed reactions since virtually all biological reactions are catalyzed by enzymes.

Enzyme Kinetics: Rates of enzyme catalyzed reactions

Usefulness of enzyme kinetics:

Common clinical assays to detect enzymes

Understanding metabolic pathways

Measuring binding of substrates and inhibitors to the active site of an enzyme

Understanding the mechanism of catalysis of an enzyme

Rates of reactions are measured by change in reactant amounts with time. You can measure the disappearance of the substrate or the appearance of the product. Usually, the appearance of the product is easier to keep track of since there should be no product present at the beginning of the reaction.

Figure 8. Ways to express a rate for the enzyme catalyzed reaction.

Rates = Reaction Velocity

For enzymes, the initial velocity (before significant product accumulates) is always used. Initial Velocity = Vo

A Simple Mechanism for the Enzyme Catalyzed Reaction.

For catalysis to begin, the substrate must bind to the enzyme, which results in the formation of the enzyme-substrate complex (ie E-S complex). The E-S complex forms rapidly in the first part of the enzyme catalysis process and the concentration of the E-S stays constant at a steady-state level. For this reason, this type of kinetics is called steady-state kinetics.

A simple mechanism for the enzyme catalyzed reaction helps us to understand and model this process.

E + S ES E + P

A simple enzyme mechanism for a single substrate and product.

Enzyme Catalyzed Rates at Different Substrate Concentrations.

Since the enzyme is used many times to catalyze the same reaction, the concentration of the enzyme is much less than the substrate:

[S] >> [E]

Thus, the substrate saturates the enzyme. This is best understood by observing the rate of the reaction or initial velocity at different [S] (ie. substrate concentrations):

[S] mM Vo mol product/min

0 0.0

1 0.9

2 1.4

5 1.9

10 2.3

50 2.6

100 2.6

Model data for the enzyme catalyzed reaction. These data show that at low [S], the initial

velocity is more or less proportional to the [S]. At high [S], the initial velocity no longer

increases as more substrate is added. Thus, at high [S] the enzyme is saturated with substrate and no increase in the enzyme catalyzed rate is observed.

This model set of data for an enzyme catalyzed reaction shows the initial velocity in terms of the amount of product formed per unit time (ie micromoles of product produced/min) at various substrate concentrations. These data can be plotted in a graphical form to also illustrate the results of an enzyme catalyzed reaction.

Plot of initial velocity of the enzyme catalyzed reaction (Vo) versus the [S] (ie

substrate concentration). Initial velocity is always given in units of amount of product formed per unit time and the substrate concentration is given in molar units (ie mM).

Here it is easy to see the saturation of the enzyme at high [S] where the initial velocity

approaches a limiting value. The plot has the shape of a square hyperbola.

The Michaelis-Menten Equation.

The plot of Vo versus [S] can be represented by an equation, which is known as the Michaelis- Menten equation in honor of the scientist who first described it. This equation, sometimes called the M-M equation, is an important one for you to know and understand.

v0 = Vmax [S ]

Km + [S ]

The Michaelis-Menten equation which describes the change in Vo as [S] increases.

The constants in this equation, Km and Vmax, are defined:

Vmax = Maximum velocity catalyzed by a fixed [E]

Km = the [S] which gives 1/2 Vmax

These definitions are illustrated below:

Vo versus [S] plot illustrating the operational definitions of Vmax and Km.

Thus, the limit approached in the Vo versus [S] plot is the Vmax.

Definition of Km and Vmax and Their Ratio - Vmax/Km.

The Km is sometimes called the Michaelis Constant. The Km is an intrinsic property of an

enzyme related to the binding constant for forming the ES complex, which is an equilibrium and can be defined by the rate constants for its formation and breakdown using the simple enzyme mechanism shown above.

The approximate relationship between the Km and the Ks for the binding of the

substrate to the enzyme which leads to the formation of the E-S complex. Ks is defined by the equilibrium formed between the enzyme (E) and substrate (S) and the E-S complex, as shown above. Ks is also defined by the ratio of the rate of breakdown of the E-S complex divided by its rate of formation.

But Km also involves the breakdown of the E-S complex to E and P, which is not a component of the Ks. Thus, the rate of the breakdown of the E-S complex to make product (P) is also defined in the simple enzyme mechanism .

The definition of Km by using rate constants for simple enzyme mechanism. The point

of this graphic is to emphasize that the Km constant of the enzyme catalyzed reaction includes more than just the formation of the E-S complex, but also its breakdown to form product, which is of course the key to an enzyme catalyzed reaction.

So Km reflects both binding of E to S but also the catalytic constant (shown as k3 above, but also defined as kcat) of the enzyme catalyzed reaction.

The Vmax is also dependent on the catalytic constant:

Vmax = kcat [E]

So both Vmax and Km are properties of individual enzymes and not very useful for comparing enzymes.

However, the ratio Vmax/Km can be used to compare enzymes. This ratio (Vmax/Km) measures the efficiency of the enzyme. The efficiency of the enzyme is ultimately limited by the rate of diffusion of the substrate to the enzyme - thus the diffusion of substrates, which is very rapid, sets an upper limit. The most efficient enzymes like Triose-P Isomerase are limited by how fast their substrates get to them. But most enzymes are not this efficient and more limited by chemical events in the active site of the enzyme.

Finding the Km and Vmax by the Graphical Solution Method.

To calculate the Km and Vmax, the Michaelis-Menten equation is converted into a linear form by taking the reciprocal of both sides of the equation. This is called the Lineweaver-Burk equation in honor of the first scientists to describe it.

The Lineweaver-Burk equation linearizes the M-M equation by taking the reciprocal of

both sides of the equation. This equation then takes on the form of the equation of a line. The y values are 1/Vo, the x values are 1/[S]. The b value in the line equation is the slope and equal to Km/Vmax, while the c value is the y-intercept and equal to 1/Vmax.

The double reciprocal plot is useful for deriving Km and Vmax by plotting kinetic data for an enzyme and you should use it to find the Km and Vmax via graphing for the problem set you got today.

The double reciprocal plot for enzyme kinetic data.

This plot must be used to find Km and Vmax for enzyme kinetic data in this class as shown on the graphic. The y-intercept is the 1/Vmax. The x-intercept, which is found in the 4th quadrant, is -1/Km. Alternatively, the Km value can be found from the slope using the Vmax value found from the y-intercept.

However, there are statistical problems with the Lineweaver-Burk equation and double

reciprocal plots, so today in research, one derives Km and Vmax using other methods such as the direct linear plot using a computer program. However, the Lineweaver-Burk equation makes the clearest representation of kinetic data and makes it easy to understand the results, so it is most often used to illustrate the data even when the Km and Vmax are derived by other methods.

Enzyme Inhibitors. A. Competitive Inhibition

Inhibitors of enzymes: Two types are considered - Competitive and Non-Competitive.

A Competitive Inhibitor has a chemical similarity to the substrate and competes with the

substrate for binding to the active site of the enzyme. A good example to describe competitive inhibition is the mitochondrial enzyme, succinate dehydrogenase:

(A) The reaction catalyzed by succinate dehydrogenase is the oxidation of succinate to fumarate. (B) Malonate and oxaloacetate are competitive inhibitors of succinate dehydrogenase.

Both these competitive inhibitors, malonate and oxaloacetate, look like succinate in their

chemical character. Both inhibitors are dicarboxylic acids like the substrate succinate so they have groups which can bind in the same places in the active site of succinate dehydrogenase as the substrate. However, neither inhibitor has the capacity to undergo the reaction and so the enzyme is inhibited. Since these inhibitors simply bind to the enzyme, when the succinate concentration is high, they will be pushed out of the site by the substrate and the enzyme will catalyze the reaction as if no inhibitor were present.

An enzyme mechanism model of the action of a competitive inhibitor (Ic) based on the standard model of a Michaelis-Menten enzyme where E + S leads to the E-S complex, which leads to product P:

Model of a Competitive Inhibitor (Ic) Interacting with the Enzyme (E) and an

equation for the equilibrium formed between the Ic and E, which is governed by the inhibitor binding constant, Ki.

This model is the same as the one described in the previous lecture where enzyme (E) and

substrate (S) bind to form the ES complex, which will go forward during catalysis to form

product (P) and the free enzyme. In the presence of the competitive inhibitor, Ic, a complex forms with enzyme when the inhibitor binds, the E-Ic complex. This is a dead-end complex and can not go on to form product. However, the Ic is bound reversibly to the enzyme and when more substrate is added, the inhibition is overcome by pulling the enzyme free via the breakdown of the E-Ic complex, which is in equilibrium with free enzyme and free Ic. Another way to think about this is - when lots of substrate is added, the concentration of free enzyme (E) falls to such a low level, that some of the E-Ic complex must breakdown to replenish the free E demanded by the equilibrium between E and Ic. This can also be demonstrated by comparing the Vo versus [S] plots for uninhibited enzyme and enzyme in the presence of a competitive inhibitor:

Vo versus [S] plot comparing the kinetics of the reaction in the absence of inhibitor and in the presence of the competitive inhibitor (Ic). At high [S], the initial velocity in the

presence of Ic will be about the same as it is in the absence of the inhibitor. The concentration of S which will be required to overcome the effect of the competitive inhibitor will depend on the [Ic] (ie. concentration of the competitive inhibitor) and the Ki (ie. the binding constant of the inhibitor to enzyme).

In competitive inhibition, addition of more substrate will out compete the inhibitor and overcome the inhibition of the enzyme's catalytic rate - thus, the Vmax will be the same and only Km will be altered. This is most clearly illustrated with the double reciprocal plot comparing the uninhibited reaction to that in the presence of Ic.

Double reciprocal plot for competitive inhibitor (Ic).

Here the uninhibited reaction gives the standard double reciprocal plot from which Km and Vmax can be calculated. The reaction in the presence of the competitive inhibitor yields apparent constants for the enzyme which are called the Km' and Vmax'. For the true competitive inhibitor, the Vmax' (apparent Vmax for inhibited enzyme) will be the same as the real Vmax, while the Km' (apparent Km for the inhibited enzyme) will be greater than the real Km. Thus, the -1/Km' will be smaller than -1/Km. After finding Km and Km', the Ki for the Ic can be calculated using the equation shown using the given concentration of the competitive inhibitor ([I]).

Enzyme Inhibitors B. Non-competitive Inhibition.

A Non-Competitive Inhibitor does not compete with substrate and the [S] has no influence on the degree of inhibition of the enzyme's catalytic rate. For example, enzymes with a thiol ( -SH ) not at the active site can be inhibited:

Example of a heavy metal inhibiting an enzyme by binding to a thiol group not at the

active site and inactivating the enzyme. Non-Competitive Inhibition can be model using the standard model for the Michaelis-Menten enzyme where E + S form the ES complex which leads to formation of product P. In this case where the non-competitive inhibitor (Inc) reacts with the enzyme at a site other than the active site, both the free enzyme (E) and the enzyme-substrate complex (E-S) react with Inc. Clearly, in this case the reaction of the non-competitive inhibitor is irreversible and the substrate can not over come the inhibitors impact on the enzyme:

Model of the Non-Competitive Inhibitor (Inc). The equilibrium between enzyme and

Inc now depends on the total concentration of enzyme in all forms present (ie. both the free E and the E-S complex) and defines the Ki.

A Vo versus [S] plot for the Non-competitive Inhibitor looks very different than that for a

competitive inhibitor since increasing the [S] has no impact:

Vo versus [S] plot for enzyme in the absence and presence of Inc.

The double reciprocal plot for this same model shows that Inc decreases Vmax, as if some of the enzyme had been removed from the system. In classic example of pure non-competitive inhibition, the uninhibited reaction and the enzyme in the presence of Inc will yield the same Km value.

Double Reciprocal plot for the Non-Competitive Inhibitor (Inc).

Non competitive inhibitors decrease Vmax but have no effect on Km.

The apparent Vmax' is smaller than the real Vmax and the Ki for the Non-Competitive Inhibitor can be calculated using the following equation and the known [I]:

Equation showing the relationship between Vmax' (apparent Vmax) and real Vmax in

the presence of a Non-Competitive Inhibitor. Use this equation for calculating the Ki of the Non- Competitive Inhibitor at known [Inc].

Evaluating Enzyme Inhibitors to determine type and their Ki.

To determine what type an inhibitor is:

1. Find Km and Vmax for uninhibited from 1/Vo vs 1/[S] plot.

2. On same graph find Km' and Vmax' for inhibited reaction.

A. If Vmax = Vmax' then inhibitor is competitive type.

(Vmax and Vmax' should not be more than 10% different)

B. If Vmax does not equal Vmax', then if Km = Km', inhibitor is non competitive type.

After finding inhibitor type, then use equations to calculate Ki. Ki is a binding constant for inhibitor to the enzyme. Ki has same units as the [I]. If [I] = mM, then Ki = mM.

Equations used for calculating Ki values:

Equation for Competitive Inhibitor.

Equation for Non-Competitive Inhibitor.

Rearrange these equations to solve for Ki.

Tutorial 3, ENV 416

1. An enzyme catalyzed reaction has a Km of 1mM and Vmax of 5 nMs-1, What is the reaction velocity when the substrate concentration is

(a) 0.25 mM

(b) 1.5 mM

2. For an enzymatic reaction, draw a plot to explain how it catalyzes a reaction.

3. (a) Differentiate between competitive inhibitor and non competitive inhibitor .

(b) Which of this is affected by change in the substrate concentration? Why?

4. Calculate Km and Vmax from the following data:

[S] (M)v0 (mM s-1)

0.1 0.34

0.2 0.53

0.4 0.74

0.8 0.91

1.6 1.04

5. Write out the enzyme mechanism model of action for competitive and non competitive inhibitor based on the MM equation.

CHAPTER 5: Nitrogen metabolism

Amino Acid Metabolism

Will be interested in two things:

1) origin of nitrogen atoms and their incorporation into amino group

2) origin of carbon skeletons

AMINO ACID SYNTHESIS

Nitrogen fixation

Gaseous nitrogen is chemically unreactive due to strong triple bond.

To reduce nitrogen gas to ammonia takes a strong enzyme --> reaction is called nitrogen fixation.

Only a few organisms are capable of fixing nitrogen and assembling amino acids from that.

Higher organisms cannot form NH4+ from atmospheric N2.

Bacteria and blue-green algae (photosynthetic procaryotes) can because they possess nitrogenase.

Enzyme has two subunits:

1) strong reductase - has Fe-S cluster that supplies e- to second subunit

2) two re-dox centers, one of which is a nitrogenase

Composed of iron and molybdenum that reduces N2 to NH4+

Reaction is ATP-dependent, but unstable in the presence of oxygen.

Enzyme is present in Rhizobium, symbiotic bacterium in roots of legumes (i.e. soybeans)

Nodules are pink inside due to presence of leghemoglobin (legume hemoglobin) that binds to oxygen to keep environment around enzyme low in oxygen (nitrogen fixation requires the absence of oxygen)

Plants and microorganisms can obtain NH3 by reducing nitrate (NO3-) and nitrite (NO2-) --> used to make amino acids, nucleotides, phospholipids.

Assimilation of Ammonia

Assimilation into amino acids occurs through glutamate and glutamine.

-amino group of most amino acids comes from -amino group of glutamate by transamination.

Glutamine contributes its side-chain nitrogen in other biosynthetic reactions.

Reaction:

NADPH +H+NADP+

NH4+ + -ketoglutarate glutamate + H2O

glutamate dehydrogenase

Another reaction that occurs in some animals is the incorporation of ammonia into glutamine via glutamine synthetase:

glutamate + NH4+ + ATP glutamine + ADP + Pi + H+

When ammonium ion is limiting, most of glutamate is made by action of both enzymes to produce the following (sum of both reactions):

NH4+ + -ketoglutarate + NADPH + ATP glutamate + NADP+ + ADP + Pi

Transamination Reactions

Having assimilated the ammonia, synthesis of nearly all amino acids is done via tranamination reactions.

Glutamate is a key intermediate in amino acid metabolism

Amino group is transferred to produce the corresponding -amino acid.

transaminase

-amino acid1-keto acid2 -keto acid1-amino acid2

Origins of Carbon Skeletons of the Amino Acids

Amino acids that must be supplied in diet are termed essential; others are nonessential.

Although the biosynthesis of specific amino acids is diverse, they all share a common feature - carbon skeletons come from intermediates of glycolysis, PPP, or citric acid cycle.

There are only six biosynthetic families:

1) Derived from oxaloacetate --> Asp, Asn, Met, Thr, Ile, Lys

2) Drived from pyruvate --> Ala, Val, Leu

3) Derived from ribose 5-phosphate --> His

4) Derived from PEP and erythrose 4-phosphate --> Phe, Tyr, Trp

5) Derived from a-ketoglutarate --> Glu, Gln, Pro, Arg

6) Derived from 3-phosphoglycerate --> Ser, Cys, Gly

Porphyrin Synthesis

First step in biosynthesis of porphyrins is condensation of glycine and succinyl CoA to form -aminolevulinate via -aminolevulinate synthase.

Translation of mRNA of this enzyme is feedback-inhibited by heme

Second step involves condensation of two molecules of -aminolevulinate to form porphobilinogen; catalyzed by -aminolevulinate dehydrase.

Third step involves condensation of four porphobilinogens to form a linear tetrapyrrole via porphobilinogen deaminase.

This is cyclized to form uroporphyrinogen III.

Subsequent reactions alter side chains and degree of saturation of porphyrin ring to form protoporphyrin IX.

Association of iron atom creates heme; iron atom transported in blood by transferrin.

Inherited or acquired disorders called porphyrias are result of deficiency in an enzyme in heme biosynthetic pathway.

congenital erythropoietic porphyria - insufficient cosynthase (cyclizes tetrapyrrole)

Lots of uroporphyrinogen I, a useless isomer are made

RBCs prematurely destroyed

Patients urine is red because of excretion of uroporphyrin I

Heme Degradation:

Old RBCs are removed from circulation and degraded by spleen.

Apoprotein part of hemoglobin is hydrolyzed into amino acids.

First step in degradation of heme group is cleavage of -methene bridge to form biliverdin, a linear tetrapyrrole; catalyzed by heme oxygenase; methene bridge released as CO.

Second step involved reduction of central methene bridge to form bilirubin; catalyzed by biliverdin reductase.

Bilirubin is complexed with serum albumin --> liver --> sugar residues added to propionate side chains.

2 glucuronates attached to bilirubin are secreted in bile.

Jaundice - yellow pigmentation in sclera of eye and in skin --> excessive bilirubin levels in blood

Caused by excessive breakdown of RBCs, impaired liver function, mechanical obstruction of bile duct.

Common in newborns as fetal hemoglobin is broken down and replaced by adult hemoglobin.

AMINO ACID CATABOLISM

Excess amino acids (those not used for protein synthesis or synthesis of other macromolecules) cannot be stored.

Surplus amino acids are used as metabolic fuel.

-amino group is removed; carbon skeleton is converted into major metabolic intermediate

Amino group converted to urea; carbon skeletons converted into acetyl CoA, acetoacetyl CoA, pyruvate, or citric acid intermediate.

Fatty acids, ketone bodies, and glucose can be formed from amino acids.

Major site of amino acid degradation is the liver.

First step is the transfer of -amino group to -ketoglutarate to form glutamate, which is oxidatively deaminated to yield NH4+ (see pathway sheet).

Some of NH4+ is consumed in biosynthesis of nitrogen compounds; most terrestrial vertebrates convert NH4+ into urea, which is then excreted (considered ureotelic).

Terrestrial reptiles and birds convert NH4+ into uric acid for excretion (considered uricotelic).

Aquatic animals excrete NH4+ (considered ammontelic).

In terrestrial vertebrates NH4+ is converted to urea via urea cycle.

One of nitrogen atoms in urea is transferred from aspartate; other is derived from NH4+; carbon atom comes from CO2.

UREA CYCLE

There are six steps of the urea cycle:

1) Bicarbonate ion, NH4+ and 2 ATP necessary to form carbamoyl phosphate via carbamoyl phosphate synthetase I (found in mitochondrial matrix).

2) Carbamoyl phosphate and ornithine (carrier or carbon and nitrogen atoms; an amino acid, but not a building block of proteins) combine to form citrulline via ornithine transcarbamoylase

3) Citruilline is transported out of mitochondrial matrix in exchange for ornithine

4) Citruilline condenses with aspartate --> arginosuccinate via an ATP-dependent reaction via arginosuccinate synthetase

5) Arginosuccinate cleaved to form fumarate and arginine via arginosuccinate lyase

fumarate --> malate--> oxaloacetate --> gluconeogenesis

oxaloacetate has four possible fates:

1) transamination to aspartate

2) conversion into glucose via gluconeogenesis

3) condensation with acetyl CoA to form citrate

4) conversion into pyruvate

6) Two -NH2 groups and terminal carbon of arginine cleaved to form ornithine and urea via arginase

Ornithine is transported into mitochondrion to repeat cycle

Overall reaction:

CO2 + NH4+ + 3 ATP + aspartate + 2 H2O ---> urea + 2 ADP + 2 Pi + AMP + PPi + fumarate

Inherited defects in urea cycle:

1) Blockage of carbamoyl phosphate synthesis leads to hyperammonemia (elevated levels of ammonia in blood)

2) argininosuccinase deficiency

Providing surplus of arginine in diet and restricting total protein intake

Nitrogen is excreted in the form of argininosuccinate

3) carbamoyl phosphate synthetase deficiency or ornithine transcarbamoylase deficiency

Excess nitrogen accumulates in glycine and glutamine; must then get rid of these amino acids

Done by supplementation with benzoate and phenylacetate (both substitute for urea in the disposal of nitrogen)

benzoate --> benzoyl CoA --> hippurate

phenylacetate --> phenylacetyl CoA --> phenylacetylglutamine

Fate of Carbon Skeleton of Amino Acids

Used to form major metabolic intermediates that can be converted into glucose or oxidized by citric acid cycle.

All 20 amino acids are funneled into seven molecules:

1) pyruvate

2) acetyl CoA

3) acetoacetyl CoA

4) -ketoglutarate

5) succinyl CoA

6) fumarate

7) oxaloacetate

Those that are degraded to acetyl CoA or acetoacetyl Coa are termed ketogenic because they give rise to ketone bodies.

Those that are degraded to pyruvate or citric acid cycle intermediates are termed glucogenic.

Leucine and lysine are only ketogenic --> cannot be converted to glucose

Isoleucine, phenylalanine, tryptophan, tyrosine are both.

All others are glucogenic only.

C3 family (alanine, serine, cysteine) ---> pyruvate

C4 family(aspartate and asparagine) ---> oxaloacetate

C5 family (glutamine, proline, arginine, histidine) ---> glutamate ---> -ketoglutarate

Methionine, isoleucine, valine, threonine --> succinyl CoA

Leucine --> acetyl CoA and acetoacetate

Phenylalanine and tyrosine --> acetoacetate and fumarate

Tryptophan --> pyruvate

Regulation of the Urea Cycle

The main allosteric enzyme is glutamate dehydrogenase.

It is inhibited by high GTP and ATP levels.

It is stimulated by high GDP and ADP levels.

Phenylketonuria

Phenylketonuria is (at least among Europeans) the most common hereditary enzyme defect. It is clinically manifest in about one among ten thousand persons. Considering that only homozygous people are clinically affected, this works out to a heterozygote frequency of (41/10,000) = 1/50,

i.e. one in fifty persons can potentially have children with this disease.

The enzyme affected is phenylalanine hydroxylase, the first enzyme in the degradative pathway . The name of the disease stems from the fact that phenylpyruvate and some derivatives thereof are found in the urine.

Formation of phenylpyruvate is due to the buildup of phenylalanine, which will eventually cause it to overcome the low KM of tyrosine transaminase .

Phenylpyruvate is believed to give rise to neurotoxic metabolites, although the exact nature of these metabolites remains to be elucidated. Symptoms include disturbances in neurological development and mental retardation.

The treatment of phenylketonuria is pretty straightforward: Limitation of dietary phenylalanine. Tyrosine is plentifully available in a modern, protein-rich diet, so that the lack of endogenous formation wont be a problem. The challenge is then to diagnose the disease in newborn kids, before any damage is done. Happily, the enzyme defect does not cause a problem during fetal development, since both useful and potentially harmful metabolites are constantly equilibrated between the maternal and the fetal circulation. Buildup of a metabolite in the fetus will therefore not occur as long as the mothers metabolism is able to degrade it.

CHAPTER 6: CARBOHYDRATE

Carbohydrates: Bountiful Sources of

Energy and Nutrients

What Are Carbohydrates?

One of the three macronutrients

Preferred energy source for the brain

Important source of energy for all cells

Composed of carbon, hydrogen, oxygen

Good sources: fruits, vegetables, and grains

Simple carbohydrates

Contain one or two molecules

Commonly referred to as sugars

Monosaccharides contain only one molecule

Glucose, Fructose, Galactose

Disaccharides contain two molecules

Lactose, Maltose, Sucrose

Complex carbohydrates

Long chains of glucose molecules

Starch, fiber, glycogen

Simple Carbohydrates Monosaccharides

Glucose Fructose Galactose

Simple Sugars Dissacharides

Complex Carbohydrates

Long chains of glucose molecules

Hundreds to thousands of molecules long

Also called polysaccharides

Starch, glycogen, most Fibers

Complex Carbohydrates

Starch

Plants store carbohydrates as starch

We digest (break down) starch to glucose

Good sources: grains, legumes, and Tubers

Glycogen

Animals store carbohydrates as glycogen

Stored in the liver and muscles

Not found in food and therefore not a source of dietary carbohydrate

Fiber

Dietary fiber is the non-digestible part of plants

Grains, seeds, legumes, fruits

Functional fiber is carbohydrate extracted from plants or manufactured

Total fiber = dietary + functional fiber

Food labels only list dietary fiber

Salivary amylase

Enzyme that begins carbohydrate digestion in the mouth

Breaks carbohydrates down to maltose

Carbohydrate digestion does not occur in the

stomach. Stomach acids inactivate salivary amylase Most chemical digestion of carbohydrates occurs in the small intestine.

Pancreatic amylase

Enzyme produced in the pancreas and secreted into the small intestine

Digests carbohydrates to maltose

Additional enzymes in the small intestine digest disaccharides to monosaccharides

Maltase breaks down maltose into two units of glucose

Sucrase breaks down sucrose into glucose & fructose

Lactase breaks down lactose into glucose & galactose

Monosaccharides are absorbed into the cells lining the small intestine and then enter the bloodstream.

All monosaccharides are converted to glucose by the liver.

Glucose circulating in the blood is our primary energy source.

Excess glucose is converted to glycogen by the LIVER

We do not have the enzymes necessary to digest fiber.

Bacteria in the large intestine can break down (ferment) some fiber. Most fiber remains undigested and is excreted in the faeces

Glucose Utilization

Blood Glucose Regulation

Blood glucose level must be closely regulated.

Hormones control blood glucose levels:

Insulin

Glucagon

Epinephrine

Norepinephrine

Cortisol

Growth hormone

Blood Glucose Regulation Insulin

Produced by beta cells of the pancreas

Stimulates glucose transporters (carrier proteins)

to help take glucose from the blood across the cell membrane

Stimulates the liver to take up glucose and convert to glycogen

Blood Glucose Regulation Glucagon

Produced by alpha cells of the pancreas

Stimulates the liver to breakdown glycogen to glucose, making glucose available to body cells Stimulates the breakdown of body proteins to amino acids to form new glucose -

Gluconeogenesis

TUTORIAL 4, ENV416/400

A. Carbohydrates

1. Name the monosaccarides produced from the hydrolysis of the dissaccharides below:

i) Sucrose

ii) Lactose

2. Explain the difference between glucose and fructose in terms of their structure.

3. D-Allosa, an aldohexose, has the same structure as D-glucose except that the carboxyl at C3 is at the plane below in the cyclic hemiacetyl form. Draw the structure of -cyclic for D-allosa.

4. Name the two component of starch. State the similarity and difference between these two structures.

5. Draw the open chain structure (Fischer projection) for D- fructose. Indicate the carbonyl ketone or aldehyde group on the molecule.

6. Draw the cyclic -D-fructose (Haworth structure).

B. Nitrogen Metabolism

1. Write the transamination reaction between -ketoglutarate and alanine.

2. Write an equation for the net reaction of the urea cycle. Show how the urea cycle is linked to the citric acid cycle.

3. Which aa in the urea cycle are the links to the citric acid cycle? Show how these links occur.

4. How many ATPs are required for one round of the urea cycle? Where do these ATPs get used?

5. What species excrete excess nitrogen as ammonia? Which ones excrete as uric acid?

CHAPTER 7 :Metabolic processes central to ATP synthesis- Glycolysis and Citric acid cycle

Glycolysis

Purpose: catabolism of glucose to provide ATPs and NADH molecules

Also provides building blocks for anabolic pathways.

Sequence of 10 enzyme-catalyzed reactions:

glucose pyruvate2 ATPs and 2 NADH produced

All enzymes (and reactions) are cytosolic.

Net reaction:

glucose + 2ADP + 2NAD+ +2Pi 2 pyruvate + 2ATP + 2NADH +2H+ +2H2O

Can catabolize sugars other than glucose:

e.g. fructose ----> 2 glyceraldehyde 3-phosphate

e.g. lactose --> glucose + galactose

galactose --> glucose 1-phosphate --> glucose 6-phosphate

e.g. mannose ---> mannose 6-phosphate --> fructose 6-phosphate

Ten Steps of Glycolysis

1) glucose --> glucose 6-phosphate by hexokinaseG = -8.0 kcal/mole

Hexokinase also works on mannose and fructose at increased [ ].

Serves to trap glucose in the cell --> a phosphorylated molecule cannot leave

2) glucose 6-phosphate --> fructose 6-phosphate by glucose 6-phosphate isomerase

Example of aldose--> ketose isomerization.

Enzyme is very stereospecific.

Reaction is near equilibrium in cell --> not a control point in glycolysis

3) fructose 6-phosphate --> fructose 1,6-bisphosphate by phosphofructokinase-1 (PFK-1)

Reaction has G = -5.3 kcal/mole and is metabolically irreversible.

Represents the first committed step in glycolysis.

4) fructose 1,6-bisphosphate --> dihydroxyacetone phosphate + glyceraldehyde 3-phosphate by fructose 1,6 bisphosphate aldolase.

5) DHAP --> glyceraldehyde 3-phosphate by triose phosphate isomerase

Also catalyzes aldose--> ketose conversion.

Rate is diffusion controlled (substrate is converted to product as fast as substrate is encountered).

6) glyceraldehyde 3-phosphate --> 1,3-bisphosphoglycerate by glyceraldehyde 3-phosphate dehydrogenase

One molecule of NAD+ is reduced to NADH --> respiratory chain

7) 1,3 bisphosphoglycerate --> 3-phosphoglycerate

Phosphoryl group transfer to ADP to form ATP.

Because phosphate group comes from a substrate molecule, called substrate level phosphorylation

First ATP-generating step of glycolysis.

8) 3-phosphoglycerate --> 2-phosphoglycerate by phosphoglycerate mutase

Mutases are enzymes that transfer phosphoryl groups from one part of a substrate molecule to another.

9) 2-phosphoglycerate --> phosphoenolpyruvate (PEP) by enolase (forms double bond)

10) PEP --> pyruvate

Second time for substrate level phosphorylation.

Reaction is metabolically irreversible.

FATE OF PYRUVATE

Under anaerobic conditions, cells must be able to regenerate NAD+ or glycolysis will stop.

Usually regenerated by oxidative phosphorylation, but that requires O2.

There are 2 anaerobic pathways that use NADH and regenerate NAD+.

1) alcoholic fermentation

Conversion of pyruvate to ethanol

H+ CO2 NADH NAD+

pyruvate acetaldehyde ethanol

pyruvate alcohol

decarboxylasedehydrogenase

glucose +2Pi + 2ADP + 2H+ ---> 2 ethanol + 2CO2 + 2ATP + 2H2O

2) lactate fermentation

NADH + H+ NAD+

pyruvate ------------------------> lactate

lactate

dehydrogenase

glucose +2Pi + 2ADP ---> 2 lactate + 2ATP + 2H20

Lactate causes muscles to ache.

Also produced by bacterial fermentation of lactose.

3) entry into citric acid cycle

The Citric Acid Cycle

Summary:

Yields reduced coenzymes (NADH and QH2) and some ATP (2).

Preparative step is oxidative decarboxylation involving coenzyme A.

Occurs in eucaryotic mitochondrion and procaryotic cytosol.

How does the pyruvate get into the mitochondrion from the cytosol?

Pyruvate passes through channel proteins called porins (can transport molecules < 10,000 daltons) located in outer mitochondrial membrane.

To get from intermembrane space to matrix involves pyruvate translocase (symporter that also moves H+ into matrix).

CONVERSION OF PYRUVATE TO ACETYL COA

Enzyme is pyruvate dehydrogenase complex, composed of three enzymes:

1) pyruvate dehydrogenase

2) dihydrolipoamide acetyltransferase

3) dihydrolipoamide dehydrogenase

Reaction occurs in 5 steps:

1) E1 uses TPP as a prosthetic group and decarboxylates pyruvate --> forms HETPP intermediate

2) E1 then transfers acetyl group to oxidized lipoamide --> acetyllipoamide

3) E2 transfers acetyl group to coenzyme A to form acetyl CoA; dihydrolipoamide becomes reduced

4) E3 reoxidizes lipoamide portion of E2; prosthetic group of E3 (FAD) oxidizes reduced lipoamide --> FADH2

5) NAD+ is reduced by E3-FADH --> E3-FAD + NADH + H+

E2 acts like a crane by swinging substrate between protein complexes in enzyme.

Regulation of PDH complex:

Regulated by covalent modification by phosphorylation.

inactive = phosphorylated; active = dephosphorylated

E1 inhibited at high [ATP]; inhibited at high [GTP]

activated by high [AMP], high [Ca2+], high [pyruvate]

E2 inhibited by high [acetyl CoA]

activated by high [CoA-SH]

E3inhibited by high [NADH]

activated by high [NAD+]

THE CITRIC ACID CYCLE

Summary:

Composed of 8 reactions

4 carbon intermediates are regenerated

2 molecules of CO2 released (6C--> 4C)

Most of energy stored as NADH and QH2

1) citrate synthase

Irreversible reaction

Acetyl CoA reacts with oxaloacetate --> citrate and CoA

2) aconitase

Citrate --> isocitrate

3) isocitrate dehydrogenase

Irreversible reaction

Substrate first oxidized (2e- and H+ given to NAD+), then decarboxylated

Isocitrate --> -ketoglutarate + CO2 + NADH + H+

4) -ketoglutarate dehydrogenase complex

-ketoglutarate first decarboxylated, oxidized (2e- and H+ given to NAD+), and HS-CoA added

Product is succinyl CoA

Enzyme complex similar the PDH, but has dihydrolipoamide succinyltransferase instead of acetyltransferase.

5) succinyl CoA synthetase or succinate thiokinase

succinyl CoA --> succinate

Substrate has high energy thioester bond; that energy is stored as nucleoside triphosphate via substrate level phosphorylation

GDP +Pi --> GTPmammals

ADP +Pi --> ATPplants and bacteria

6) succinate dehydrogenase complex

Enzyme is embedded in inner mitochondrial membrane.

Has FAD covalently bound to it (prosthetic group).

Converts succinate --> fumarate with generation of FADH2 --> ETS

FAD is regenerated by reduction of a mobile molecule called ubiquinone (coenzyme Q) --> QH2.

CHAPTER 8 :Electron Transport and Oxidative Phosphorylation

Oxidative phosphorylation - process in which NADH and QH2 are oxidized and ATP is produced.

Enzymes are found in inner mitochondrial membrane in eukaryotes.

In prokaryotes, enzymes are found in cell membrane.

Process consists of 2 separate, but coupled processes:

1) respiratory electron-transport chain

Responsible for NADH and QH2 oxidation

Final e- acceptor is molecular oxygen

Energy generated from electron transfer is used to pump H+ into intermembrane space from matrix ---> matrix becomes more alkaline and negatively charged.

2) ATP synthesis

Proton concentration gradients represents stored energy

When H+ are moved back across inner mitochondrial membrane through ATP synthase ---> ADP is phosphorylated to form ATP

Chemiosmotic Theory of ATP Production

Proposed by Peter Mitchell in 1961 (won Nobel Prize for this work).

Proton concentration gradient serves as energy reservoir for ATP synthesis.

Proton concentration gradient also known as proton motive force (PMF).

Components of Electron Transport System

There are 5 protein complexes:

I) NADH-ubiquinone oxidoreductase

II) succinate-ubiquinone oxidoreductase

III) ubiquinol-cytochrome c oxidoreductase

IV) cytochrome c oxidase

V) ATP synthase

Electrons flow through ETS in direction of increasing reduction potential.

Two mobile electron carriers also involved: ubiquinone (Q) between complexes I or II and III, and cytochrome c between complexes III and IV.

Electrons enter ETS 2 at a time from either NADH or succinate.

I - NADH-ubiquinone oxidoreductase

Transfers 2e- from NADH to Q as hydride ion (H-)

First electron transferred to FMN --> FMNH2 ---> Fe-S cluster ---> Q

Also pumps 4H+/2e- into intermembrane space

II - succinate-ubiquinone oxidoreductase

Transfers e- from succinate to Q

First transferred to FAD ---> FADH2 ---> 3 Fe-S clusters ---> Q

Not enough energy to contribute to proton gradient via proton pumping

III - ubiquinol-cytochrome c oxidoreductase

Rransfers e- from QH2 to cytochrome c facing intermembrane space

Composed of 9-10 subunits including 2 Fe-S clusters, cytochrome b560, cytochrome b566, and cytochrome c1.

Transports 2H+ from matrix into intermembrane space

IV - cytochrome c oxidase

Contains cytochromes a and a3

Contributes to proton gradient in two ways:

1) pumps 2H+ for each pair of e- transferred (per O2 reduced)

2) consumes 2H+ when oxygen is reduced to H2O ---> lowers [H+]matrix

Carbon monoxide (CO) and cyanide (HCN) bind here

V - ATP synthase

Does not contribute to H+ gradient, but helps relieve it

Also called FOF1 ATP synthase

F1 component contains catalytic subunits

FO component is proton channel that is transmembrane

Per ATP synthesized, 3H+ move through ATP synthase

oligomycin - antibiotic that binds to channel and prevents proton entry --> no ATP synthesized

TRANSPORT OF MOLECULES ACROSS MITOCHONDRIAL MEMBRANE

Inner mitochondrial membrane is impermeable to NADH and NAD+.

Must use a shuttle to regenerate NAD+ for glycolysis; solution is to shuttle electrons across membrane, rather than NADH itself.

There are two shuttles in operation:

1) glycerol phosphate shuttle

Found in insect flight muscles and mammalian cells in which high rates of oxidative phosphorylation must occur

Cytosolic glycerol 3-phosphate dehydrogenase converts DHAP to glycerol 3-phosphate

Converted back to DHAP by membrane-bound glycerol 3-phosphate dehydrogenase

Result is transfer to 2e- to FAD --> Q ---> complex III

Produces fewer ATP molecules (1.5 vs. 2) because complex I is bypassed

2) malate-aspartate shuttle

Found in liver and heart

Cytosolic NADH reduces oxaloacetate --> malate --> transported via dicarboxylate translocase into matrix

In matrix, malate --> oxaloacetate --> aspartate ---> transported out via glutamate-aspartate translocase

Converted back to oxaloacetate.......

No reduction in ATP yield

Must also be able to transport other metabolites into and out of matrix:

1) ADP/ATP carrier or ADP/ATP translocase

Adenine nucleotide translocase which exchanges ADP and ATP (antiporter)

2) Pi/H+ carrier

Couples inward movement of Pi with symport of H+ from gradient

REGULATION OF OXIDATIVE PHOSPHORYLATION

Depends upon substrate availability and energy demands in the cell.

Important substrates are NADH, O2, and ADP.

As ATP is used, more ADP is available, translocated through adenine nucleotide translocase --> electron transport increases.

Known as respiratory control.

Helps to replenish ATP pool in the cell, which is kept nearly constant.

Rates of glycolysis, citric acid cycle, and electron transport system are matched to a cells ATP requirements.

Proton gradient can be short-circuited to generate heat

Found in brown adipose tissue in newborn mammals and animals that hibernate, and animals adapted to cold conditions

A protein called thermogenin forms a proton channel in inner mitochondrial membrane --> dissipates proton gradient, but electrons still flow --> heat production

Pathway is activated by fatty acids from triacylglycerol catabolism from epinephrine stimulation

Superoxide Production

Even though cytochrome oxidase and other proteins that reduce oxygen have been designed not to release O2.- (superoxide anion), it still does happen.

Protonation of superoxide anion yields hydroperoxyl radical (HO2.), which can react with another molecule to produce H2O2.

Enzyme superoxide dismutase catalyzes this reaction

2H+

O2.- + O2.- ----------------------------> H2O2 + O2

superoxide dismutase

Recent findings have indicated that superoxide dismutase mutations can cause amyotrophic lateral sclerosis (Lou Gehrigs disease), in which motor neurons in brain and spinal cord degenerate.

The hydrogen peroxide formed is scavenged by catalase:

H2O2 + H2O2 2H2O + O2

catalase

Peroxidases catalyze an analogous reaction:

ROOH + AH2 ROH + H2O + A

peroxidase

7) fumarase

fumarate --> malate

8) malate dehydrogenase

L-malate --> oxaloacetate

2e- and H+ given to NAD+ --> NADH

Net reaction for citric acid cycle:

acetyl CoA + 3NAD+ + Q + GDP(ADP)+ Pi +2H2O ---> HS-CoA + 3NADH + QH2 + GTP(ATP) + 2CO2 + 2H+

Energy Budget so far from 1 molecule of glucose:

glycolysis2 ATP2 NADH

Prep Step2 NADH

TCA2 ATP6 NADH2 QH2

4 ATP 10 NADH

ATP Production:

glycolysis2 ATP6 ATP equivalents

Prep Step6 ATP equivalents

TCA2 ATP18 ATP equivalents + 4 ATP equivalents

4 ATP34 ATP = 38 ATPs maximum

substrate(ox. phos.)

level phos.

REGULATION OF TCA CYCLE

There are 2 enzymes that are regulated:

1) isocitrate dehydrogenase

allosterically activated by high [Ca2+] and high [ADP]

allosterically inhibited by high [NADH]

2) -ketoglutarate dehydrogenase

allosterically activated by high [Ca2+]

allosterically inhibited by high [NADH] and high [succinyl CoA]

ENTRY AND EXIT OF METABOLITES

Citrate, -ketoglutarate, succinyl CoA, oxaloacetate lead to biosynthetic pathways.

Citrate --> fatty acids and sterols in liver and adipocytes

(cleaved into acetyl CoA if needed)

-ketoglutarate --> glutamate --> amino acid synthesis or nucleotide synthesis

succinyl CoA --> propionyl CoA --> fatty acid synthesis

--> porphyrin synthesis

oxaloacetate --> gluconeogenesis

--> asparate --> urea synthesis, a.a. synthesis, pyrimidine synthesis

Pathway intermediates must be replenished by anapleurotic reactions.

GLYOXYLATE CYCLE

Modification of citric acid cycle.

Anabolic pathway in plants, bacteria, yeast.

Takes 2 carbon compounds and converts them to glucose.

Common in plants which store energy reserves as oils, but must be converted to carbohydrates during germination.

In eucaryotes, a glyoxysome is a special organelle where this occurs.

Gluconeogenesis, the Pentose Phosphate Pathway and Glycogen Metabolism

GLYCOGEN METABOLISM

Glycogen stored in muscle and liver cells.

Important in maintaining blood glucose levels.

Glycogen structure: 1,4 glycosidic linkages with 1,6 bran