Chapter 7 Enzyme Mechanism & Control (1)

8/3/2019 Chapter 7 Enzyme Mechanism & Control (1)

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Paul D. Adams University of Arkansas

Mary K. Campbell

Shawn O. Farrellhttp://academic.cengage.com/chemistry/campbell

Chapter SevenThe Behavior of Proteins:

Enzymes, Mechanisms, and Control
http://academic.cengage.com/chemistry/campbellhttp://academic.cengage.com/chemistry/campbell


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Allosteric Enzymes

Allosteric: Greek allo+ steric, other shape

Allosteric enzyme: an oligomer whose biological activity is affected byother substances binding to it

these substances change the enzymes activity by altering the

conformation(s) of its 4structure

Allosteric effector: a substance that modifies the behavior of an allosteric

enzyme; may be an

allosteric inhibitor

allosteric activator

Aspartate transcarbamoylase (ATCase)

feedback inhibition


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Feedback Inhibition

Formation of product

inhibits its continuedproduction


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ATCase

Rate of ATCase catalysis vs

substrate concentration

Sigmoidal shape of curve describes

allosteric behavior

ATCase catalysis in presenceof CTP; ATP


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ATCase (Contd)

Organization of ATCase

catalytic unit: 6 subunitsorganized into 2 trimers

regulatory unit: 6 subunitsorganized into 3 dimers

Catalytic subunits can beseparated from regulatorysubunits by a compound thatreacts with cysteine (p-hydroxymercuribenzoate)


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Allosteric Enzymes (Contd)

Two types of allosteric enzyme systems exist

Note: for an allosteric enzyme, the substrateconcentration at one-half Vmax is called the K0.5

K system: an enzyme for which an inhibitor oractivators alters K0.5

V system: an enzyme for which an inhibitor oractivator alters Vmax but not K0.5


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Allosteric Enzymes (Contd)

The key to allosteric behavior is the existence of multiple

forms for the 4structure of the enzyme allosteric effector: a substance that modifies the 4

structure of an allosteric enzyme

homotropic effects: allosteric interactions that occur

when several identical molecules are bound to theprotein; e.g., the binding of aspartate to ATCase

heterotropic effects: allosteric interactions that occurwhen different substances are bound to the protein;e.g., inhibition of ATCase by CTP and activation byATP


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The Concerted Model

Wyman, Monod, and Changeux - 1965

The enzyme has two conformations

R (relaxed): binds substrate tightly; the active form

T (tight or taut): binds substrate less tightly; the

inactive form in the absence of substrate, most enzyme molecules

are in the T (inactive) form

the presence of substrate shifts the equilibrium from

the T (inactive) form to the R (active) form in changing from T to R and vice versa, all subunits

change conformation simultaneously; all changes areconcerted


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Concerted Model (Contd)

A model represented by a protein having two conformations

Active (R) form-Relaxed binds substrate tightly, Inactive (T) form-Tight (taut) binds substrate less tightly both change from T to R atthe same time

Also called the concerted model

Substrate binding shifts equilib. To the relaxed state.

Any unbound R is removed KR


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The model explains the sigmoidal effects

Higher L means higher favorability of free T form

Higher c means higher affinity between S and R form,more sigmoidal as well.


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An allosteric activator (A) binds to and stabilizes the R

(active) form An allosteric inhibitor (I) binds to and stabilizes the T

(inactive) form

Effect ofbindingactivatorsand inhibitors


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Sequential Model (Contd)

Main Feature of Model:

the binding of substrate induces a conformationalchange from the T form to the R form

the change in conformation is induced by the fit of thesubstrate to the enzyme, as per the induced-fit modelof substrate binding

sequential model represents cooperativity


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Sequential Model (Contd)

Sequential model for cooperative binding of substrate to an allosteric enzyme

R form is favored by allosteric activator

Allosteric inhibition also occurs by the induced-fit mechanism

Unique feature of Sequential Model of behavior:

Negative cooperativity- Induced conformational changes that make the enzyme

less likely to bind more molecules of the same type.

Sequential Model:


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Control of Enzyme Activity via Phosphorylation

The side chain -OH groups

of Ser, Thr, and Tyr canform phosphate esters

Phosphorylation by ATP can

convert an inactiveprecursor into an activeenzyme

Membrane transport is acommon example


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Membrane Transport

Source of PO4 is ATP

When ATP is hydrolyzed, energy released that allows other

energetically unfavorable reactions to take place

PO4 is donated to residue in protein by protein kinases


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Zymogens

Zymogen: Inactive precursor of an enzyme where cleavage

of one or more covalent bonds transforms it into the activeenzyme

Chymotrypsinogen

synthesized and stored in the pancreas

a single polypeptide chain of 245 amino acid residuescross linked by five disulfide (-S-S-) bonds

when secreted into the small intestine, the digestiveenzyme trypsin cleaves a 15 unit polypeptide from the N-terminal end to give -chymotrypsin


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Activation of chymotrypsin

Activation of chymotrypsinogen by proteolysis


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Chymotrypsin

A15-unit polypeptide remains bound to -chymotrypsin by

a single disulfide bond -chymotrypsin catalyzes the hydrolysis of two dipeptide

fragments to give -chymotrypsin

-chymotrypsin consists of three polypeptide chains joined

by two of the five original disulfide bonds changes in 1structure that accompany the change from

chymotrypsinogen to -chymotrypsin result in changes in

2- and 3structure as well.

-chymotrypsin is enzymatically active because of its 2

-and 3structure, just as chymotrypsinogen was inactivebecause of its 2- and 3structure


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The Active Site

Some important questions to ask about enzyme mode of action:

Which amino acid residues on an enzyme are in the active siteand catalyze the reaction?

What is the spatial relationship of the essential amino acidsresidues in the active site?

What is the mechanism by which the essential amino acidresidues catalyze the reaction?

As a model, we consider chymotrypsin, an enzyme of thedigestive system that catalyzes the selective hydrolysis ofpeptide bonds in which the carboxyl group is contributed byPhe or Tyr


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Kinetics of Chymotrypsin Reaction

p-nitrophenyl acetate is

hydrolyzed bychymotrypsin in 2stages.

At the end of stage 1,

the p-nitrophenolate ionis released.

At stage 2, acyl-enzymeintermediate ishydrolyzed and acetate(Product) isreleasedfree enzyme

is regenerated


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Chymotrypsin

Reaction with a model substrate


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Chymotrypsin (Contd)

Chymotrypsin is a serine protease

DIPF inactivates chymotrypsin by reacting withserine-195, verifying that this residue is at the active

site


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H57 also critical for

activation of enzyme

Can be chemically

labeled by TPCK


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Because Ser-195 and His-57 are required for activity,

they must be close to each other in the active site

Results of x-ray crystallography show the definitearrangement of amino acids at the active site

In addition to His-57 and Ser-195, Asp-102 is alsoinvolved in catalysis at the active site

The folding of the chymotrypsin backbone, mostly inantiparallel pleated sheet array, positions the essential aminoacids around the active-site pocket


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The active site of

chymotrypsin showsproximity of 2 reactivea.a.

M h i f A ti f C iti l A i A id i


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Mechanism of Action of Critical Amino Acids inChymotrypsin

Serine oxygen is nucleophile

Attacks carbonyl group of peptide bond


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Catalytic Mechanisms

General acid-base catalysis: depends on donation

and acceptance of protons (proton transfer reactions)

Nucleophilic substitution catalysts- Nucleophilicelectron-rich atom attacks electron deficient atom.

same type of chemistry can occur at active site of

enzyme: SN1, SN2


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Catalytic Mechanisms (Contd)

Lewis acid/base reactions

Lewis acid: an electron pair acceptor

Lewis base: an electron pair donor

Lewis acids such as Mn2+, Mg2+, and Zn2+ are essentialcomponents of many enzymes (metal ion catalysts)

carboxypeptidase A requires Zn2+ for activity


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Catalytic Mechanisms (Contd)

Zn2+ of

carboxypeptidase iscomplexed with:

The imidazole sidechains of His-69 andHis-196 and the

carboxylate sidechain of Glu-72

Activates the

carbonyl group fornucleophilic acylsubstitution


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Enzyme Specificity

Absolute specificity: catalyzes the reaction of one unique

substrate to a particular product

Relative specificity: catalyzes the reaction of structurallyrelated substrates to give structurally related products

Stereospecificity: catalyzes a reaction in which onestereoisomer is reacted or formed in preference to all othersthat might be reacted or formed

example: hydration of a cis alkene (but not its transisomer) to give an R alcohol (but not the S alcohol)


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Asymmetric binding

Enzymes can be

stereospecific(Specificity whereoptical activity may paya role)

Binding sites on enzymes

must be asymmetric


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Active Sites and Transition States

Enzyme catalysis

an enzyme provides an alternative pathway with a loweractivation energy

the transition state often has a different shape than either thesubstrate(s) or the product(s)

True nature of transition state is a chemical species that is

intermediate in structure between the substrate and the product. Transition state analog: a substance whose shape mimics that of atransition state

In 1969 Jenks proposed that

an immunogen would elicit an antibody with catalytic activity if

the immunogen mimicked the transition state of the reaction the first catalytic antibody or abzyme was created in 1986 by

Lerner and Schultz

*(Biochemical Connections, p. 196)


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Coenzymes

Coenzyme: a nonprotein substance that takes part in an

enzymatic reaction and is regenerated for further reaction metal ions- can behave as coordination compounds. (Zn2+,

Fe2+)

organic compounds, many of which are vitamins or are

metabolically related to vitamins (Table 7.1).


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NAD+/NADH

Nicotinamide adenine

dinucleotide (NAD+

) is usedin many redox reactions inbiology.

Contains:1) nicotinamide ring

2) Adenine ring

3) 2 sugar-phosphate groups


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NAD+/NADH (Contd)

NAD+ is a two-electron oxidizing agent, and is

reduced to NADH

Nicotinamide ring is where reduction-oxidation

occurs


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B6 Vitamins

The B6 vitamins are coenzymes involved in amino group

transfer from one molecule to another. Important in amino acid biosynthesis


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Pyridoxal Phosphate

Pyridoxal and pyridoxamine phosphates are involved in

the transfer of amino groups in a reaction calledtransamination

Figure 7.21 p. 197

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Chapter 7 Enzyme Mechanism & Control (1)