Lecture 14 - Enzymology

Characteristics of enzymes

1) biological catalysts

2) not consumed during a chemical reaction

3) speed up reactions from 1000 - 1017, with a mean increase in rate of 100,000

4) exhibit stereospecificity --> act on a single stereoisomer of a substrate

5) exhibit reaction specificity --> no waste or side reactions

Nomenclature

Typically add “-ase” to name of substrate

e.g. lactase breaks down lactose (dissacharide of glucose and galactose)

IUBMB classifies enzymes based upon the class of organic chemical reaction catalyzed:

1) oxidoreductase - catalyze redox reactions

dehydrogenases, oxidases, peroxidases, reductases

2) transferases - catalyze group transfer reactions; often require coenzymes

3) hydrolases - catalyze hydrolysis reactions

4) lyases - lysis of substrate; produce contains double bond

5) isomerases - catalyze structural changes; isomerization

6) ligases - ligation or joining of two substrates with input of energy, usually from ATP

hydrolysis; often called synthetases or synthases

Enzyme kinetics

A mathematical and graphical study of the rates of enzyme-catalyzed reactions.

k

S -----------> P

k

A + B ---> C

The velocity of this reaction can be summarized by the following equation:

v = k[S] or v = k[A][B]

This reaction is considered a first-order reaction, determined by the sum of the exponents in the

rate equation --> number of molecules reacting.

There are also bimolecular reactions, which involve two substrates; good example of group transfer

reactions.

S1 + S2 ---> P1 + P2

v = k[S1][S2] first order for each reactant;

but second order overall

For enzyme-catalyzed reactions:

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E + S -----> ES -----> E + P

The rate or velocity is dependent upon both [enzyme] and [substrate].

In reality, enzyme-catalysed reactions are not that simple:

k1 kcat

E + S ES E + P

k-1

k1 and k-1 govern the rates of association and dissociation of ES

kcat is the turnover number or catalytic constant

VES = k1[E][S]

VE+S = k-1[ES]

VE+P = kcat[ES]

Usually an enzyme’s velocity is measured under initial conditions of [S] and [P].

These same reactions can be described graphically:

velocity

[S]

At low [S], vo increases as [S] increases.

At high [S], enzymes become saturated with substrates, and the reaction is independent of

[S] --> display saturation kinetics.

Vmax = kcat[ES]

or because the [S] is irrelevant at high [S]

Vmax = kcat [E]

The graph is a graph of a hyperbola, and the equation for a hyperbola is

y = ax

b + x where a is the asymptote

b is value at a/2

Substituting our equation parameters,

Vo = Vmax[S]

Km + [S] Michaelis-Menten equation

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Different enzymes reach Vmax at different [S] because enzymes differ in their affinity for the

substrate or Km.

1) The greater the tendency for an enzyme and substrate to form an ES, the higher the

enzyme’s affinity for the substrate ---> lower Km.

2) At a given [S], the more enzyme will be in ES for an enzyme with a higher affinity

i.e. the greater the affinity, the lower the [S] needed to saturate the enzyme or to

reach Vmax.

Enzyme-substrate affinity and reaction kinetics are closely associated

[S] at which vo=1/2Vmax = Km

Km is a measure of enzyme affinity

Km = k-1

k1 reflection of association and dissociation of

ES

a small Km (high affinity) favors E + S ----> ES

a large Km (low affinity) favors ES -----> E + S

meaning that the lower the Km, the less substrate is needed to saturate the enzyme.

We would like numbers of Vmax and Km for a means of comparison among enzymes.

It is difficult to estimate Vmax and Km from a typical graph of [substrate] vs. velocity.

These two parameters are used to describe the efficiency of enzymes; must be an easier method

for measuring these parameters.

Done by transformation of the date by taking the reciprocal of both sides of the equation --->

double reciprocal plot or Lineweaver-Burke plot.

Vo = Vmax[S]

Km + [S]

1 Km 1 1

Vo = Vmax [S] + Vmax y=mx+b

Alterations in enzyme activity:

Enzyme inhibition

Molecule that binds to enzyme and interferes with its activity to prevent either:

1) formation of ES complex E + I ---> EI

2) breakdown of ES --> E + P ES + I ---> ESI

Used to regulate metabolism.

Many drugs act by enzyme inhibition.

These molecules can be

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1) irreversible - bind to enzymes by covalent means and modify enzyme

2) reversible - noncovalent binding to enzyme

There are three types of reversible inhibition:

1) competitive

Competes with substrate for active site of enzyme.

Both substrate and competitive inhibitor bind to active site.

These inhibitors are often substrate analogs (similar in structure substrate), but

still no product is formed.

Can be overcome by addition of more substrate (overwhelm inhibitor; a numbers

game).

e.g. malonate inhibition of succinate dehydrogenase

succinate ----------------------> fumarate

FAD FADH2

succinate malonate

e.g. AZT inhibition of HIV reverse transcriptase

actual substrate is dTTP (deoxythymidine triphosphate)

Can be represented by the following equation:

E + S ES E + P

+

I

EI

Graphical representation of competitive inhibitors:

affects Km (increases Km --> decreases affinity; need more substrate to reach half-

saturation of enzyme)

Vmax unaffected

2) uncompetitive inhibitor

Typically seen in multisubstrate reactions (here, there is a decrease in product

formation because the second substrate cannot bind).

Inhibitor binds to ES, but not enzyme.

E + S ES E + P

+

I

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ESI

Graphical representation of uncompetitive inhibitors:

Lineweaver-Burke plot:

both Km and Vmax are lowered, usually the same amount

ratio Km/Vmax unchanged --> no change in slope

3) pure noncompetitive inhibitor

Can bind to enzyme and ES complex equally.

Does not bind to same site as substrate and is not a substrate analog.

Cannot be overcome by increases in [substrate].

e.g. lead, mercury, silver, heavy metals

Lineweaver-Burke plot:

No effect on Km, because those enzyme molecules unaffected have normal

affinity.

Vmax is lowered.

Regulation of Enzyme Activity There are many ways to regulate enzyme activity at different levels:

1) regulation of rate of synthesis or degradation

Is fairly slow (several hours), so is really too slow to be effective in eukaryotic cells.

Need something that can occur in seconds or less.

Usually done through regulatory enzymes and occur in metabolic pathways early or

at first committed step:

E1

A + B ---> C ---> D ---> E --->F ---> P feedback inhibition

G ---> H

Result is to conserve material and energy by preventing accumulation of

intermediates.

2) allosteric regulation

Done through allosteric sites or regulatory sites on enzymes - site other than active

site where inhibitor or activator can bind.

Properties of allosteric enzymes:

1) sensitive to metabolic inhibitors and activators

2) binding is noncovalent; not chemically altered by enzyme

3) regulatory enzymes possess quaternary structure - individual

polypeptide chains may or may not be identical

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4) enzyme has at least one substrate that gives sigmoidal curve

due to positive cooperativity because of multiple substrate

binding sites.

Theories of allosteric regulation:

1) concerted theory or symmetry-driven theory

Assumes 1 binding site/subunit for each ligand.

Enzyme can assume either R or T conformation.

Assumes that all subunits are in R or T state, and all switch at same

time when the first substrate is bound.

2) sequential theory

Ligand introduces a change in the tertiary structure of a

subunit.

Only that subunit is converted to R conformation.

3) covalent modification

Usually requires one enzyme to activate enzyme and another to inactivate.

Most common modification is phosphorylation of serine residues on interconvertible

enzyme (the one that does the activating).

e.g. pyruvate dehydrogenase

Enzyme Mechanisms

All enzymatic reactions go through a transition state (unstable intermediate form with a structure

between that of reactant and product).

Reactants must collide precisely to form transition state.

Must have correct orientation.

Must collide with enough energy = activation energy = G

Enzymes work by lowering G

Substrates are correctly oriented. All increase probability of reaction.

Transition states are stabilized.

Chemical Catalysis

Active site of most enzymes is lined with hydrophobic amino acids.

There are a few polar a.a. which make up the catalytic center of the active site and can be ionized.

Histidine (basic a.a.) is common.

Aspartate and glutamate - negatively charged

Lysine and arginine - positively charged; electrostatic binding can occur

There are several types of chemical catalysis:

1) acid-base catalysis

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Enzymes that use this have a.a. side chains that can donate or accept electrons to

substrate.

Can accelerate a chemical reaction by a factor of 10-100.

2) covalent catalysis

Substrate forms a covalent bond with enzymes, then part of substrate is

transferred to a second substrate in a 2 step process.

A-X + E X-E + A

X-E + B B-X + E

3) proximity effect

Collection of substrate molecules in the active site increases the concentration

over those molecules found freely in solution.

Result is that there is a more frequent formation of transition states.

4) transition state stabilization

Increase binding of transition states to enzymes compared to substrate or

product alone binding.

Binding forces are charge-charge interactions, hydrogen bonds, hydrophobic

interactions, van der Waals forces.

Often seen in side chains of aspartate, glutamate, histidine, lysine, and arginine.

pH affects enzymatic rates:

Inflection points approximate pKa of ionizable residues important in active site.

e.g. papain

Examples of enzyme mechanisms:

1) triose phosphate isomerase

Catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde

3-phosphate.

Enzyme has two ionizable active site residues Glu (glutamate)-165 and His-95--> act as acid-

base catalysts.

Mechanism:

1) Hydrogen bonds form between imidazole group of His-95 and carbonyl oxygen of DHAP

(COO- group is ionized; histidine active a.a.)

Carboxylate group of Glu-195 attacks proton of C-1 from substrate (DHAP) to form

enediolate intermediate.

2) C-2 oxygen (electron-rich) attacks proton of His-95 --> converts -O- to -OH to form an

enediol

3) imidazole from of His-95 attacks -OH of C-1 to form another enediolate intermediate

4) Glu-195 donates proton to C-2 --> glyceraldehyde 3-phosphate formed

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2) chymotrypsin, a member of the serine proteases

Examples: trypsin, chymotrypsin, and elastase that catalyze much of the digestion in the

small intestine.

Synthesized in pancreas; stored as inactive precursors called zymogens - prevents damage

to cell.

What is actually released from the cell is trypsinogen, chymotrypsinogen, proelastase, which

are then activated by selective proteolysis.

Enzyme called enteropeptidase activates trypsinogen --> trypsin by cleaving of N-terminal

hexapeptide.

Trypsin then activates the other two.

All three enzymes have similar primary, second, tertiary structure.

All cleave peptide bonds on COOH side of hydrophobic or aromatic side chains.

Substrate specificity is due to amino acid residues in the hydrophobic binding pocket.

o chymotrypsin - serine (uncharged) --> accepts large, bulky, hydrophobic

side groups.

o trypsin - aspartate ( negatively charged) accepts Lys, Arg, Gly, Ala.

o elastase - shallow - binds a.a. with small side chains ( Gly, Ala).

Catalysis involves use of catalytic triad:

o Ser-195 His-57 Asp-102

All three amino acids are hydrogen bonded.

Ser-195 residue is highly reactive; very unusual.

Mechansim of chymotrypsin:

1) Substrate enters enzyme and is aligned with R1 group in binding pocket --> places carbonyl

carbon of peptide bond next to oxygen of Ser- 195.

2) His-57 attacks H of Ser-195.

3) Now, the nucleophilic oxygen of Ser-195 attacks carbonyl carbon of peptide bond to form

tetrahedral intermediate (transition state?).

4) C=O bonds changes to a single bond (oxygen is negatively charged = oxyanion) and forms H-

bond with -NH groups of Gly-193 and Ser-195.

5) His-57 and Asp-102 share H+ in low-barrier hydrogen bond (increases rate of catalysis by

decreasing activation energy; very strong hydrogen bond).

6) His-57 imidazolium ring acts as an acid catalyst by donating H to peptide bond --> molecule

cleaved --> amine product released.

7) Carbonyl group of peptide forms covalent bond with enzyme --> acyl-enzyme intermediate

formed.

8) After first product leaves, a molecule of water enters --> donates H+ to His-57 --> -OH

group left attacks carbonyl group --> formation of second tetrahedral intermediate and

stabilized by oxyanion hole plus a low barrier H-bond.

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9) His-57 donates a proton --> second intermediate collapses

10) Second product is formed, released from active site --> chymotrypsin regenerated.

Shows: covalent catalysis (Ser), acid-base catalysis (His)