23_april_enzyme_mechanism_and_regulation

8/7/2019 23_April_Enzyme_Mechanism_and_Regulation

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What Are the Mechanisms of Enzyme-Induced Rated Accelerations?

Mechanisms of catalysis:

Entropy loss in ES formation

Destabilization of ES

Covalent catalysis

General acid/base catalysis

Metal ion catalysis

Proximity and orientation

Why Is the Binding Energy of ES Crucialto Cataylsis?

Competing effects determine the position ofES on the energy scale

Try to mentally decompose the bindingeffects at the active site into favorable andunfavorable

The binding of S to E must be favorable

But not too favorable!

Km cannot be "too tight" - goal is to makethe energy barrier between ES and EX

small


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What Roles Do Entropy Loss and Destabilizationof the ES Complex Play?

Raising the energy of ES raises the rate

For a given energy of EX, raising the energyof ES will increase the catalyzed rate

This is accomplished by

a) loss of entropy due to formation of ES

b) destabilization of ES by

strain

distortion desolvation

Formation of the ES complex results in a loss of entropy. Prior to binding, E and Sare free to undergo translational and rotational motion. By comparison, the EScomplex is a more highly ordered, low-entropy complex.


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Substrates typically lose waters of hydration in the formationof the ES complex. Desolvation raises the energy of the ES

complex, making it more reactive.

Electrostatic destabilization of a substrate may arise from juxtaposition of like

charges in the active site. If such charge repulsion is relieved in the course ofthe reaction, electrostatic destabilization can result in a rate increase.


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What Are the Mechanisms ofCatalysis?

Serine Proteases are good examples ofCovalent catalysis

Enzyme and substrate become linked in acovalent bond at one or more points in thereaction pathway

The formation of the covalent bond provideschemistry that speeds the reaction

General Acid-base Catalysis

Catalysis in which a proton is transferred in thetransition state

"Specific" acid-base catalysis involves H+ orOH- that diffuses into the catalytic center

"General" acid-base catalysis involves acidsand bases other than H+ and OH-

These other acids and bases facilitatetransfer of H+ in the transition state


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Catalysis of p-nitrophenylacetatehydrolysis by imidazolean exampleof general base catalysis. Proton

transfer to imidazole in the transitionstate facilitates hydroxyl attack on thesubstrate carbonyl carbon.

Liver alcohol dehydrogenasecatalyzes the transfer of ahydride ion ( H:-) from NADHto acetaldehyde (CH3CHO),forming ethanol (CH3CH2OH).An active-site zinc ion

stabilizes negative chargedevelopment on the oxygenatom of acetaldehyde, leadingto an induced partial positivecharge on the carbonyl Catom. Transfer of thenegatively charged hydride ionto this carbon forms ethanol.


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An example of proximity effects in catalysis. (a) Theimidazole-catalyzed hydrolysis of p-nitrophenylacetate isslow, but (b) the corresponding intramolecular reaction is

24-fold faster (assuming [imidazole] = 1 Min [a]).

What Can Be Learned from TypicalEnzyme Mechanisms?

Serine proteases and aspartic proteases aregood examples

Knowledge of the tertiary structure of anenzyme is important

Enzymes are the catalytic machines that sustainlife


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The Serine Proteases

Trypsin, chymotrypsin, elastase, thrombin,subtilisin, plasmin, TPA

All involve a serine in catalysis - thus the name

Ser is part of a "catalytic triad" of Ser, His, Asp

Serine proteases are homologous, but locationsof the three crucial residues differ somewhat

Enzymologists agree, however, to number themalways as His-57, Asp-102, Ser-195

Comparison of theamino acid sequencesof chymotrypsinogen,trypsinogen, and

elastase. Each circlerepresents one aminoacid. Numbering isbased on the sequenceof chymotrypsinogen.

Filled circles indicateresidues that areidentical in all threeproteins. Disulfidebonds are indicated inyellow. The positions ofthe three catalytically

important active-siteresidues (His57, Asp102,and Ser195) are

indicated.


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Serine Protease Mechanism

A mixture of covalent and general acid-basecatalysis

Asp-102 functions only to orient His-57 His-57 acts as a general acid and base Ser-195 forms a covalent bond with

peptide to be cleaved Covalent bond formation turns a trigonal C

into a tetrahedral C The tetrahedral oxyanion intermediate is

stabilized by N-Hs of Gly-193 and Ser-195

The catalytic triad

of chymotrypsin .


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The substrate-binding pockets of trypsin, chymotrypsin, andelastase. (Illustration: Irving Geis. Rights owned by Howard Hughes MedicalInstitute. Not to be reproduced without permission. )

Diisopropylfluorophosphate (DIFP) reacts with active-site serine residues ofserine proteases (and esterases), causing permanent inactivation.


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A detailed mechanism for the

chymotrypsin reaction. Note thelow-barrier hydrogen bond(LBHB) in (c) and (g).


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The oxyanion

hole of

chymotrypsinstabilizes thetetrahedraloxyaniontransition statesof the mechanism

What Factors Influence EnzymaticActivity?

Six points:

Rate slows as product accumulates

Rate depends on substrate availability

Genetic controls - induction and repression

Enzymes can be modified covalently

Allosteric effectors may be important Zymogens, isozymes and modulator

proteins may play a role


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Enzymes regulated by covalent modification are called interconvertible enzymes. The

enzymes (protein kinaseand protein phosphatase, in the example shown here) catalyzingthe conversion of the interconvertible enzyme between its two forms are called converterenzymes. In this example, the free enzyme form is catalytically active, whereas the

phosphoryl-enzyme form represents an inactive state. The -OH on the interconvertibleenzyme represents an -OH group on a specific amino acid side chain in the protein (for

example, a particular Ser residue) capable of accepting the phosphoryl group.

Proinsulin is an 86-residue precursor toinsulin (the sequenceshown here is humanproinsulin). Proteolytic

removal of residues 31to 65 yields insulin.Residues 1 through 30(the B chain) remainlinked to residues 66through 87 (the A chain)by a pair of interchain

disulfide bridges.


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The proteolytic activation of chymotrypsinogen.

The cascade ofactivation steps leadingto blood clotting. Theintrinsic and extrinsicpathways converge at

Factor X, and the finalcommon pathwayinvolves the activation ofthrombin and itsconversion of fibrinogeninto fibrin, whichaggregates into ordered

filamentous arrays thatbecome cross-linked toform the clot.


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The isozymes of lactate dehydrogenase (LDH). Active muscle tissue becomes anaerobicand produces pyruvate from glucose via glycolysis. It needs LDH to regenerate NAD+ fromNADH so glycolysis can continue. The lactate produced is released into the blood. Themuscle LDH isozyme (A4) works best in the NAD

+-regenerating direction. Heart tissue isaerobic and uses lactate as a fuel, converting it to pyruvate via LDH and using the pyruvateto fuel the citric acid cycle to obtain energy. The heart LDH isozyme (B4) is inhibited byexcess pyruvate so the fuel wont be wasted.

What Are the General Features of AllostericRegulation?

Action at "another site" Enzymes situated at key steps in metabolic

pathways are modulated by allostericeffectors

These effectors are usually producedelsewhere in the pathway

Effectors may be feed-forward activators orfeedback inhibitors

Kinetics are sigmoid ("S-shaped")


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Sigmoid vversus [S] plot. The dotted line represents the hyperbolic plot characteristicof normal Michaelis - Menten-type enzyme kinetics.

Cyclic AMP- dependent protein kinase (also known as PKA) is a 150- to 170-kD R2C2tetramer in mammalian cells. The two R (regulatory) subunits bind cAMP (KD = 3 x 10

-8

M); cAMP binding releases the R subunits from the C (catalytic) subunits. C subunits areenzymatically active as monomers.


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Glycogen Phosphorylase

Allosteric Regulation and CovalentModification

Pi is a positive homotropic effector

ATP is a feedback inhibitor, and a negativeheterotropic effector

Glucose-6-P is a negative heterotropiceffector (i.e., an inhibitor)

AMP is a positive heterotropic effector (i.e.,

an activator)

The mechanism ofcovalent modificationand allosteric regulationof glycogenphosphorylase. The Tstates are blue and theR states blue-green.


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In this diagram of the glycogen phosphorylasedimer, the phosphorylation site (Ser14) and theallosteric (AMP) site face the viewer. Access tothe catalytic site is from the opposite side of theprotein. The diagram shows the major

conformational change that occurs in the N-terminal residues upon phosphorylation ofSer14. The solid black line shows theconformation of residues 10 to 23 in the b, orunphosphorylated, form of glycogen

phosphorylase. The conformational change inthe location of residues 10 to 23 uponphosphorylation of Ser14 to give the a(phosphorylated) form of glycogenphosphorylase is shown in yellow. Note thatthese residues move from intrasubunit contactsinto intersubunit contacts at the subunitinterface. [Sites on the two respective subunitsare denoted, with those of the upper subunit

designated by primes ().](Adapted from Johnson, L. N., and Barford, D., 1993.The effects of phosphorylation on the structure andfunction of proteins. Annual Review of Biophysics andBiomolecular Structure 22:199-232.)

Regulation of GP by CovalentModification

In 1956, Edwin Krebs and Edmond Fischershowed that a converting enzyme couldconvert phosphorylase b to phosphorylase a

Three years later, Krebs and Fischer showthat this conversion involves covalentphosphorylation

This phosphorylation is mediated by anenzyme cascade


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The hormone-activated enzymatic cascade that leads to activation of glycogenphosphorylase.

23_april_enzyme_mechanism_and_regulation

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