Enzymes Are Bio Catalysts Formed in Cells Either as Simple or as Conjugated Proteins With a Non

8/3/2019 Enzymes Are Bio Catalysts Formed in Cells Either as Simple or as Conjugated Proteins With a Non

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Enzymes are biocatalysts formed in cells either as simple or as conjugated proteins with a non-amino acidcomponent.

Coenzymes often function in the transfer of electrons or of functional groups (hydrogen atom, acetyl, methyl,amino groups, etc.). The coenzymes are generally identical with vitamins which, at least in higher organisms, representessential components of their food that cannot be synthesized in their organs.

The enzymes accelerate biological reactions by decreasing the activation energy of a given reaction withoutaltering its equilibrium. The mechanism of their action consists in the formation of a complex between enzyme andsubstrate (ES) which undergoes the chemical reaction proper whereupon the enzyme-product complex (EP) is splitto the original enzyme and the product. The existence of some enzymesubstrate complexes has now been proved

both indirectly (spetrophotometry) and directly (chemical isolation).

The substrate is bound to the enzyme in the active site or centre which may be visualized as a spatial arrange-ment of certain amino acids of the protein moiety of the enzyme as well as of the prosthetic group or the coenzyme.Amino acids frequently found to play a role in the active site are serine (through its OH group) and histidine (throughthe nitrogen of its imidazole). Some enzymes occur in the active form directly upon their synthesis while others aresynthesized as inactive proenzymes (pepsinogen, trypsinogen) or exist a part of their life-time in an inactive conformation(allosteric enzymes). They must then be activated by a special process to become functional.

Enzyme-catalyzed reactions proceed at different rates, depending on(a) the amount or activity of the enzyme,(b) the concentration of substrate,(c) the pH and composition of the solution,(d) temperature,(e) the presence of activators and inhibitors.

a. Since the enzyme concentrations in living cells are difficult to estimate we often speak about their activities.Enzyme activity is measured in international units (U), corresponding to an activity converting 1 (1 substrate

per min, or, more recently, in katals (kat), corresponding to aiactivity converting 1 mol substrate per s. Specificactivity of an isolated enzyme is expressed in units per mg protein. Specificity of an enzyme defines the rangeof structure types the enzyme can attack. If the specificity is absolute the enzyme can catalyze the chemical reactionof a single compound, if it is relative, several related compounds can serve as substrate. Stereospecificity indicatesthat the enzyme accepts only a certain stereo-isomer (L- or D-form). A given substrate can be converted by one ofseveral theoretically possible reactions catalyzed by different enzymes. This is important in some metabolic control

processes.b. At low substrate concentrations the enzyme reaction follows first-order kinetics, i.e. the rate is proportional

to substrate concentration. At very high concentrations of substrate the reaction is ofzero order, i.e. the enzyme issaturated by its substrate. Every enzyme reaction has its characteristic Michaelis constant,, which defines theconcentration at which the enzyme reaction proceeds at half its maximum rate. It is not identical with the dissociationconstant of the ES complex just as its reciprocal is not identical with the association constant of this complex.

Every enzyme-catalyzed reaction has its pH optimum.d. Every enzyme-catalyzed reaction proceeds most rapidly at a certain temperature.

e. Activators and inhibitors are compounds that can bear on the rate of the enzyme reaction either positively ornegatively. Activators increase the enzyme reaction by aiding the formation of a functional active site and are mostoften represented by metal ions, such as Mg2+, Zn2+, Mn2+, Co2+. Activation ofallosteric enzymes which arecomposed of subunits is of paramount importance in the control of multi-enzyme systems; this is usually achieved

by various organic molecules.

Enzyme inhibitors can be of several kinds, the most important being competitive and noncompetitive ones.Competitive inhibitors interact with the active site of the enzyme and resemble the substrate in its structure so thatthey compete with it for binding. They may be removed by excess substrate. Noncompetitive inhibitors react withanother important structure of the enzyme molecule (e.g., a SH group). The inhibition can be either reversible orirreversible in the case that the inhibitor brings about a permanent (covalent) change of an essential functional groupof the enzyme. A noncompetitive inhibitor cannot be removed by excess substrate.

To distinguish between the various types of inhibition a number of kinetic and graphical techniques can be used(e.g., that of Lineweaver and Burk).

Underin vivo conditions (in cells) most enzymes are spatially organized in the so-called multi-enzyme systems.These are either bound to cell structures or are freely dissolved in various cell compartments. Their intracellular

concentrations are usually higher than those of the available intermediates (millimolar with most metabolites).

Enzymes are named after the reaction they catalyze, by attaching the suffix -ase, and are divided into six mainclasses. Both systematic and trivial enzyme names are in use.


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Nature of group transferred Name of coenzyme Name of enzyme

(code number)

Function

(examples of coenzyme

dependent rections)Free Bond on coenzyme

ATP Hexokinase

(2.7.1.1)Phosphate transfer (to

and from OR.)

Hexoses

phosphorylation

-" ATP Ri bosophosphatepyrophosphatekinase(pyrophosphotransferase)(2 7.16)

Pyrophosphate transfer

Phosphorylation

of D-riboso-5-phosphateto 5-phosphonbosyl-1 -pyrophosphate

-"- ATP ArmnoacyJ-tRNA ,synthetases (Itgases)

(6.1.1.4 to 6.1 1.21)

Adenylate transfer(to and

from RCO) ll Amino

acids activation

-"- ATP Methyl adenosyltransferase (2.5.1 6)Adenosyl transferto and from methionine

GTP Phosphoenol

pyruvate carboxy

kinase (4.1.1.32)

Formation

of phosphoenol pyruvate

from oxatoacetate (Some

phosphate transfer

reactions.)

GDP Mannose 1-

phosphate guanilyl

tranferase (2 7.7.13)

Some carbohydrate i

ntercon verstons

(mannose -+ fucose)

ITP. Phosphoenolpyruvate carboxykinase (41 1 32)

See GTP

UDP Uridyltransferases

(2.7.7.9 to 2.7.712)

Carbohydrate

mterconversions,

oxidations, isomerisations,

synthesis

CTP Cyti dy .transferases

(2.781, 2.7.714; 2.7 7

15)

Phospholipid

intercon versions

NADH Dehydrogenases

(pyridine linked)

(111.1 to 11.1.45)

Reversible transfer of two

reducing equivalents from

substrate to the coenzyme

oxidized form

NADPH Dehydrogenases(pyridine linked)(1.1.1.10; 1.1119; 1 11 34; 1.1 1 36;1.11.49)

Carrier of energy-rich

electrons. Transfer from

catabolic reactions to

electron requiring anabolic

reactions Synthesis of

some hydrogen -rich

biomolecufes

/ADH2 Dehydrogenases

(flavin linked)

(1.3.99.1, 1.3.99.2)

Direct transfer

of two hydrogen atoms

from substrate

to the oxidized form

of coenzyme (oxidative

deamination of amino

acids)

FMNH, Dehyd rogenases

(fiavine linked)

See FADH2

TPP

(thiamine

pyrophosphate)

Tranai do lase

(2.2.1 2)

TransketoUse (2

2.1 1)

Transfer

of "active aldehyde"

(glycoialdehyde,

acetaldehyde)and dihydroxyetny!group


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)

a) =0

b) ,

)

Pyridoxal

phosphate

Transaminase

aminotransferase)

(2.6.1.1. to 2.6.1-52)

decarboxylase

amino acid)

[4.1.1.22) acemase

Transformation

of ct-L-amino acids to:

a-ketoaci ds

amines

oc-D-amino acids

Biotin (N-

carboxylate)

Carboxylase

(6.4.1.1 to

6.4.1.3)

Carbon-dioxld transfer.

[Pyruvate carboxylase,

acetyl CoA carboxylase,

propionyl CoA

carboxylase)

N10 formy! FH*

10~formyl-5. 6, 7, 8-

tetrahydro folic

acid

Phosphoribosyi

amino-Imidazol-

carboxamid formyl

transferase (2.1.2.3)

Formyl transfer.

Biosynthesis of purine

nucleotides

N* formyl FH4 5-

formyl-5, 6. 7, o-

tetrahydrofoltc acid

For my (transferase

(2.1.2.6).

Formyl transfer.

Biosynthesis of purine

nucleotides

N**10methenyl FH*

5,10-methenyl-5, 6,

7,8-tetrahydrofollc

acid

Phosphori bosy I

-glyeinamid formyl

transferase (2.1.2.2)

Formyl transfer.

Biosynthesis of

purine nucleotides

N5 formimino FbU 5-

formimino-5, 6, 7, 8-

tetrahydrofolic

add

Glutamate

formimino

transferase

(2.1.2.5)

Transfer

of formimino group from

amino acids (Gly, Glu)

N5*10 methylen FH

5,IO-methylen-5. 6.

7,8-tetra-hydrofolic

acid

Thy m idyl ate

synthetase

Methyfation of dUMP

todTMP

N* methyl FH4 5-

methyl-5, 6, 7,

8-tetrahydrofolie acid

Methyltransferase Methyfationof homocysteineto methionine


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Co bam ide Methylmalonyl-CoA

mutase

(5.4.99.2)

Rearrangement

and synthesis

of methyl group.

Isomer is ation

of methylmalonyl CoA

to succinyl CoA,glutamic acidto ^-methylaspartic aci

CoA-SH

Coenzyme A

Acetyltransferase(2.3.1.1to 2.3.1.12)Acyltransferase(2.3.1.15 . to2.3.1.20)

Reactions

of carboxylic acids

(including acetate).

Biosynthesis

of carboxylic acids

S-aeyl li poate

reduced

Pyruvatedehydrogenase(1.2.4.1)Ketogluurate

dehydrogenase

(1.2.4.2)

Transfer of !

residues generated by

a-keto acids

decarboxylation

PAPS

Phosphoadenosine

phosphosulfate

Sulfotransferase

(2.8.2.1 to 2.8.2.5)

Transfer of sulfate to

phenol, steroids,

arylamine, chondroitine

Coenzyme Function Correspondingvitamin

Pyridoxal phosphate TransaminationDecarboxylationRaoemization

Pyridoxine (Be)

Thiamine

pyrophosphate

Aerobic decarboxy-

lationTransfer of thealdehyde group

Thiamine (Bi)

Coenzyme A Transfer of acylsAerobic degradationand synthesis of fattyacids

Pantothenic acid

Tetrahydrofolicacid

Transfer of carbongroups

Folic acid

Biotin Transfer of C02 Biotin (H)

NAD + Transfer of H+ + e- Nicotinic acid (niacin)()

NADP+ Transfer of H+ + e- Nicotinic acid (PP)

FMN Transfer of H+ + e- Riboflavin (Ba)

FAD Transfer of H+ + e- Riboflavin ()

C O E N Z Y M E S A N I? R E L A T Si V ST

AM i N S

It is one of the characteristics of coenzymes thathigher organisms cannot synthesize them and,therefore, they must be supplied with food. Becausetheir function is solely catalytic the daily requirementis low (several mg per day in man).

Compounds known as vitamins in human dietare in most cases identical with or closely related to

coenzymes. This fact underlies the importance ofvitamins in nutrition physiology. If vitamins arenot in ample supply, metabolic disturbances mayoccur, known as hypovitaminoses and avitaminoses.


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E F F E C T OF pH ON E N Z Y M E

A C T I V I T Y

Most enzymes are characterized by their pHdependence, there being a certain pH value of opti-mum enzyme activity. On both sides of this valuethe activity is lower. The optimum pH values range

widely from highly acidic (pepsin) to highly alkaline(alkaline phosphatase). Therefore, in all enzymestudies, the pH must be maintained by a suitablebuffer.

The pH dependence of enzyme activity is deter-mined by the pKof ionizable groups of the enzymemolecule, particularly those at or near the active site(possibly playing a role in the binding of the coenzy-me), and those that can contribute to-changes of theactive site through conformational changes of partsof the protein molecules. The pH can further affectthe degree of ionization or the spatial organization ofsubstrate (including proteins). Most pronounced arethe differences in the pH optimum in the digestive

tract.

E F F E C T OF T E M P E R A T U R E ON ~ N! XY H

A C T I V I T Y

Temperature always affects the reaction rate.Within the physiological range, the reaction rate willincrease with temperature (1) but above a certain point enzyme-catalyzed reactions are affected byheat denaturation of the enzyme protein molecule (2).This results in a dependence with an optimum.

Not all enzymes are affected by temperatureidentically. This can be used for the separation ofsome specific enzymes. The activity of an enzyme

mixture is estimated at a lower temperature, that ofthe more stable component is estimated at a raisedtemperature.

A C T I V A T I O N Of- E N Z Y M E S

1. By cleavage of an oligopeptide from a proenzyme.2. By formation of SS bonds, resulting in the

exposure of the active site (activation ofribonuclease by subtilisin).

3. By formation of a complex with metal ions.4. By allosteric activation.


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E N Z Y M E ACTIVATION AND

F O R M A T I O N O F T H E A C T I V E

SITE l

The case ofchymotrypsin is used to demonstratethese concepts. Under the action of pepsin or auto-catalytically, two dipeptides are split from the origi-

nally single polypeptide chain with 249 amino acidsof chymotrypsinogen (Ser14-Arg15 and Thr147--Asn148)which gives rise to three peptide chainsinterconnected by five SS bridges. Thisnecessarily changes the conformation of the moleculeand an active site of the enzyme is formed. Chymo-trypsin has thus been formed from chymotrypsino-gen-

The active site is formed by Ser1'95, His87 andAsp1"2.

A similar molecule, including the amino acidsequence of some parts of the protein chain, is thatof trypsinogen (239 amino acids). It is activated totrypsin by splitting off a hexapeptide from the amino

end of the chain. The conformation of the trypsinmolecule is stabilized by six SS bonds whichlink together a single polypeptide chain.

A L L O S T E R I C E N Z Y M E S

are oligomers formed by two or more monomers.They can exist in two extreme, reversible states,called active and inactive. Every ligand (substrate,effector) that can form a complex with the proteincan bind to each of the protein subunits: to an activesite for the substrate, and to a regulatory site for theeffector.

Binding of the effector to a subunit is followed bya gradual change of conformation of other subunits.This results in a gradual transition of the allostericenzyme to the active state. If the active state ischanged back to the inactive one, the binding site forsubstrate changes its activity.

As the protein passes from one conformation to theother, its molecular symmetry is preserved.

Allosteric enzymes participate in the control ofmetabolism in multi-enzyme systems. Thecontrol of a metabolic sequence is effected by a ne-gative or positive feedback.

1. States of an allosteric enzyme caused by achange of spatial organization.

2. Allosteric activation through binding an acti-vator (positive effector).

3. Allosteric inhibition due to binding an inhibitor(negative effector).


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MECHANISM OF E N Z Y M E ACTION

Mechanism of acetylcholine cleavage by cho-linesterase

The active site of the enzyme contains two functionallyand spatially separated sites:

1. COO- where the positively charged N+ of thesubstrate is bound electrostatically j

2. the active site proper possessing an esterase activity,with Ser, , His.

The substrate acetylcholine is bound in the reaction in adefinite position in the_ active site by an ionic bondbetween the negative charge of COO-of the active site andthe positively charged quaternary nitrogen N+= of thesubstrate.

During the reaction, a proton dissociated from thephenol group of (active site) combines with the oxygenof the originally alcoholic group of choline. This generatesa positive charge at the carbon of the substrate acetyl group

which is attracted to the negatively charged oxygen of thedissociated alcoholic group of serine in the active site. Thebond between (choline) and (acetyl) is broken and theliberated acetyl reacts with serine to a transientacetylserine. The proton split off from serine is attracted tothe negatively charged oxygen of the dissociated phenolgroup of tyrosine, a hydroxy group is formed and theoriginal state of the tyrosine residue of the active site isrestored. The hydrolysis proper begins by dissociation of awater molecule, the H+ being attracted to the nitrogen ofthe imidazole group of His, the hydroxyl is set free andattacks the transiently formed ester bond of acetyl-serine.This liberates a molecule of acetic acid. The H+ boundtransiently to the imidazole nitrogen is set free and re-

attached to the O-

of serine. The original state of all thethree active-site amino acids is restored. The releasedcholine and acetic acid leave the active centre by diffusion.

All the processes described above take place more orless simultaneously. The hydrolysis of acetylcholineresults from a combined action of all the functional groupsof the active site.

The reaction course is affected by the distances betweenthe individual subsites in the active centre. Modelexperiments done with substrate analogs indicate that thereaction will proceed smoothly if the basic group ofimidazole lies 0.5 am from the COO- group of acetyl, andif the tyrosine chain lies 0.25 nm from the side chain ofserine. This provides further evidence for the importance

of the active site conformation for its activity.


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K I N E T I C S OF E N Z Y M E -CATALYZED R E A C T I O N S

At a given enzyme concentration the reaction ratedepends on the concentration of substrate. This substratedependence of the rate is graphically described by arectangular hyperbola such that at low concentrations of

substrate the reaction is first-order while at very highsubstrate concentrations it is zero-order.This observation was the basis for the formulation of a

fundamental theory of enzyme kinetics, by L. Michaelisand M. L. Menten in 1913. It is based on the assumptionthat an enzyme-substrate complex is formed whichundergoes a chemical reaction and breaks down to the freesnzyme and a product (eq. 1). A characteristic value ofevery enzyme reaction, the, can be derived as follows.

The actual enzyme concentration after the complex hasbeen formed is [E] = [E(] [ES]. The overall reaction ratedepends on the rates at which the ES complex is formedand broken down. The re-formation of the ES complexfrom the enzyme and the product is usually neglected. Atsteady state, the rate of ES formation vi and the rate of itsdecomposition 2 are identical and thus expression 2 maybe used. The rate of product formation is given by v &a[ES]. The value of [ES] can be computed from eq. 3 andsubstituted in eq. 5.

The maximum rate Kmax is attained at high substrateconcentrations when all the enzyme molecules aresaturated. Then [ES] = [Ej] and eq. 6 can be used. Eq. 5can now be rearranged by USmg max- The KM can beestimated from measurable quantities, substrateconcentration, reaction rate, and maximum reaction rate(eq. 7). Ifv =

* , eq. 8 can be applied, showing that

has the dimension of concentration.

TheKM. value depends on the type of substrate, the pHof the reaction mixture and the temperature. If an enzymecatalyzes the reaction of several related substrates, everysubstrate will have its characteristic KM- In anapproximation, reactions with a lowerKM will proceedmore readily. '

L I N E A R I Z A T I O N Of THEM I C H A E L S EQUATiOK (Lineweaver and Burk)

The reaction rate v is expressed by eq 9. If a reciprocalof the equation is taken, it has the form of (10) and, afterrearrangement, of (11) which is an equation of the straightline

= a + bx where = 1/v; a =1/Kmax (this determines the intercept with the y-axis); b =/ (this is the slope of the straight line); 1/[S].


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C O M P E T I T I V E I N H I B I T I O N

is reversible and can be relieved by a high con-centration of substrate. It is made possible by a lowerspecificity of the binding site of the enzyme so thatstructurally related compounds can be bound but not

necessarily chemically transformed. The reactionrate depends on the ratio of inhibitor and substrateconcentrations and on their relative affinity for theenzyme. The apparent value increases.

N O N C O M P E T IT I V E I N H I B I T I O N

cannot be relieved by excess substrate. The in-hibitor binds allotopically with respect to substrateand is not structurally related to the substrate. Thereaction rate depends on the concentration ofinhibitor and on the inhibition constant Ki. Theapparent Fmai decreases as (1 + [l]lKi) rises.

A L L O S T E R I C I N H I B I T I O N

is either reversible or irreversible. It is caused by anegative effector (inhibitor) being bound at a site

different from the active site (a regulatory site). Theinhibitor is often the final reaction product of amulti-enzyme system. The dependence of thereaction rate on the concentrations of substrate andinhibitor is not simple and cannot be expressed as achange of- This is connected with the fact thatthe dependence of initial rate on substrateconcentration in allosteric enzymes usually has an S-shaped character.


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Clan Subgroup Reaction catalyzed

1. Oxidoreductases hydrogenation and dehydrogena-tion

1.11.2

1.31.41.5

1.6

2. Transferases transfer of functional groups

2.12.22.32.4

2.52.62.72.8

one-carbon residues aldehyde orketo group acylglycosyl bond nonmethyl alkylor aryl nitrogen-containing

group phosphorus-containinggroups sulfur-cntaining groups

3. Hydrolases hydrolytic reactions of

3.13.23.33.43.53.6

estersglycosidesetherspeptidesother C-N bondsacid anhydrides

4. Lyases addition to a double bond

4.1

4.2

4.3

5. Isomerases isomerizations

5.15.25.35.4

racemases and epimerases cis-traru isomerases intramolecularoxidoreductases intromaleculartransferases

6. Ligases bond formation using ATP

6.16.2

6.3

6.4

E N Z Y M E C L A S S I F I C A T I O N

When coining names of enzymes, the substratename was usually taken and the suffix -ase wasattached (arginase catalyzes the hydrolysis of argi-nine; phosphatase hydro lyzes phosphoric esters,etc.). In other cases, the suffix was attached to the

name of the catalyzed reaction, thus: dehydrogenasecatalyzes dehydrogenations, hydrolase catalyzeshydrolysis, transferase a transfer of a chemical group,etc. Some of the early described enzymes have specialnames, such as trypsin, pepsin, catalase.

As the number of enzymes known keeps increasing,the International Union of Biochemistry recommen-ded to introduce a decimal system of enzymes basedon the nature of the catalyzed reaction. Such a clas-sification provides direct information on the characterof the catalyzed process. In 1972, the Commissionfor Biochemical Nomenclature of the InternationalUnion of Pure and Applied Chemistry (IUPAC)published a new edition of Enzyme Nomenclature

(1973).The first number in the classification describe the

main class (2 stands for Transferases), the nextnumber refers to some characteristic of the reaction(2.1 indicates the transfer of a one-carbon residue),the next number provides further details (2.1.1means transfer of a methyl group). Both systematicand trivial (but logically constructed) names are nowin use.


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OXIDOREDUCTASES

Several types of enzyme-catalyzed oxidation-reduction reactions. The enzymes transferhydrogenor electrons and catalyze biological oxidations.They contain specific coenzymes. They are groupedaccording to the donor from which they accept

hydrogen or electron, or according to the acceptor towhich they transmit it.

Enzyme Group t ransferred General react ion

Phosphor ransf erase Phosphoryl H1PO4

Aminotransferase Amino NHj

Sulfotransferase Sulfuryl -SO3H

(transferase Acetyl, succinyl.aminoacyl

TRANSFERASES

transfer groups of atoms by specific carrierswhich act as coenzymes. They play a role in bio-chemical conversions and can transfer methyl,carboxyl, amino, sulfo, formyl (Ci) or phosphorylgroups.

Enzyme Substrate Bond attached General reaction

Peptidases ProteinsPeptides

Peptide

Glycoside

hydrolasesPolysaccharides

disaccharides

Glycoside

Esterases

Lipases

Neutral lipids

phospholipids

Ester

Phospho-

diesterases

Polynucleotide Phosphodlester

Phosphatases

Phosphate

esters

Ph os ph noes te*

H Y D R O L A S E S

catalyze' hydrolytic cleavage and are namedaccording to the bond they attack (glycosidases,esterases, etc.).


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Enzyme Group removed General reaction

Decarboxylase CO,

Aldol&se

Lyase or synthase for

reverie reaction

Ketoicid

Dehydratase HiO

Deaminase NH3

LYASES

are enzymes splitting groups from the substratemolecule nonhydrolytically; also they form doublebonds or assist in the addition of groups to double bonds. They can split off carbon dioxide, water,ammonia, and larger groups.

Enzyme Group

isomerteed

Alternative

position of group

General reaction

Glucose 6-phosphate

fsomerase

Carb onyl C-1 ------- -* C-2

Phosphoglycerate

phospho mutase

Phosphoryl C -2

Documents

Enzymes Are Bio Catalysts Formed in Cells Either as Simple or as Conjugated Proteins With a Non