Vous êtes sur la page 1sur 112

CHAPTER 11 Mechanism of Enzyme Action

1. General properties of enzymes 2. Activation energy and the reaction coordinate 3. Catalytic mechanism 4. Lysozyme 5. Serine proteases

Enzyme act with great speed and precision

1. Enormous variety of chemical reactions within a cell 2. Mediated by Enzymes 3. Enzymology, the study of enzymes (coined 1878; Greek: en, in; zyme, yeast), fermentation: glucose -> ethanol 12 enzyme-catalyzed steps 4. James Summer, 1926, crystallized urease from jack bean, shown to be a protein 5. Other catalysts, i.e. ribozymes (peptide-bond formation; RNA-world), only for units 6. Proteins more versatile, 20 functional units

Introduction Enzymes increase the rate of chemical reactions by lowering the free energy barrier that separates the reactants and products

1. General Properties of Enzymes

Enzymes differ from ordinary chemical catalysts by: - Higher reaction rates, 106-1012 - Milder reaction conditions (temp, pH, ) - Greater reaction specificity (no side products) - Capacity for regulation

Definition catalyst: catalyzes reaction but is not itself consumed during the process

Table 11-1

A) Classification of Enzymes
- naming: -ase, urease, alcohol dehydrogenase but no rules, - systematic: IUBMB: 6 Classes acc. to the nature of the chemical reaction that is catalyzed (http://expasy.org/enzyme/)

B) Enzymes Act on Specific Substrates

- Noncovalent forces through which substrates bind to enzymes: van der Waals, electrostatic, hydrogen bonding, hydrophobic intercations - Geometric Complementarity - Electronic Complementarity - Induced fit upon substrate binding - lock-and-key model (proposed by Emil Fischer)

An Enzyme-Substrate complex
Geometric and electrostatic complementarity

Enzymes are Stereospecific

- Enzymes are highly specific both in binding to chiral substrates and in catalyzing stereo-specific reactions - Enzymes are themselves are chiral, L-amino acids -> active centers = active site is asymmetric/ stereo selective

Citrate is prochiral and is stereospecifically transformed into isocitrate

Stereospecificity in substrate binding

Enzymes vary in geometric Specificity

- Stereoselectivity, right hand into left glove - Geometric specificity is a more stringent requirement than stereoselectivity, old key into modern lock: i.e. alcohol dehydrogenase, oxidation of ethanol (CH3CH2OH) to acetaldehyde (CH3CHO) faster than methanol to formaldehyde or isopropanol to aceton even though they only differ by deletion or addition of one CH2 group !

Some enzymes are very permissive, chymotrypsin, can hydrolyze amide and ester bonds, exception rather than rule !

Some Enzymes Require Cofactors

- Can act as enzymes *chemical teeth to take over chemical reactions that cannot be performed by amino acid side chains - Required in diet of organisms - for example metal ions, Cu2+, Fe3+, Zn2+ toxicity, Cd2+ and Hg2+ can replace Zn and inactivate the enzyme - organic molecules, coenzymes, can transiently associate with enzyme as cosubstrate, i.e., nicotinamide adenine dinucleotide (NAD+)

Types of Cofactors in Enzymes

The structure and reaction of NAD+

NAD+ is an obligatory cofactor in The alcohol dehydrogenase (ADH) reaction

NADH dissociates from the enzyme to be re-oxidized in an independent reaction

Prosthetic groups

Permanently associated with enzyme, often by covalent bonds, example heme is bound to proteins called cytochromes Holoenzyme = enzyme+cofactor complex, active Apoenzyme, lacks cofactor, inactive

Coenzymes must be regenerated

In order to complete the catalytic cycle, the coenzyme must return to its original state i.e. by a different enzyme such as is the case with NADH

2) Activation Energy and the Reaction Coordinate

Transition State Theory: developed in 1930s HA-HB + HC -> HA + HB-HC Transition state: HA--HBHC Transition state = point of highest free energy = most unstable
Reactants approach one another along a path of minimal free energy = reaction coordinate Transition state diagram/reaction coordinate diagram: Plot of free energy versus the reaction coordinate

Transition State Diagram (Symetrical)

Transition State



Transition State Diagram (Asymetrical)

Free energy of activation

Free energy of reaction

Activation Energy and the Reaction Coordinate

The greater the free energy of activation, the slower the reaction rate If the free energy of the reaction, G<0, then the reaction is spontaneous and releases energy (heat)

Transition State Diagram For a Two-Step Reaction



Catalysts Reduce the free energy of activation, G

Catalysts act by providing a reaction pathway with a transition state whose free energy is lower than that of the uncatalyzed reaction

Effect of a catalyst on the transition state diagram of a reaction

Catalysts Reduce the free energy of activation, G

Reaction rate is proportional to e-G/RT G of 5.7kJ/mol (1/2 of one hydrogen bond) gives 10-fold rate enhancement G of 34kJ/mol (small fraction of a covalent bond) give 106-fold enhancement

Note: the catalyst enhances rate of forward and that of the back reaction by the same magnitude, but Greaction determines whether forward or back reaction is favored

3) Catalytic Mechanisms
Enzymes lower the free energy of the transition state (G) by stabilizing the transition state
Learn about enzymatic reactions mechanisms by examining the corresponding non-enzymatic reactions of model compounds

Catalytic Mechanisms
Curved arrow convention to trace electron pairs At all times, rules of chemical reasons apply to the system, i.e. never five bonds on C, or 2 on H etc.

Types of Catalytic Mechanisms

1. Acid-base catalysis 2. Covalent catalysis 3. Metal ion catalysis 4. Proximity and orientation effects 5. Preferential binding of the transition state

A) Acid-Base Catalysis occurs by Proton Transfer

General acid catalysis: Proton transfer from an acid lowers the free energy of a reactions transition state Example, keto-enol tautomerization (a) Enhanced by proton donation (b) or proton abstraction (c) (general base catalyzed)

Concerted Acid-Base Catalysis

Asp, Glu, His, Cys, Tyr, Lys have pKs in or near the physiological range The ability of enzymes to arrange several catalytic groups around their substrates makes concerted acid-base catalysis a common enzymatic mechanism

Effects of pH on Enzyme Activity

Most enzymes are active only within a narrow pH range of 5-9. Reaction rates exhibit bell-shaped curves in dependence of pH (reflects ionization state of important residues) pH optimum gives information about catalytically important residues, if 4/5 -> Glu, Asp; 6->His, 10->Lys pK of residues can vary depending on chemical environment +/- 2

pH Optimum of Fumarase

RNase A is an acid-base catalyst

Bovine pancreatic RNase A: Digestive enzyme secreted by pancreas into the small intestine 2,3 cyclic nucleotides isolated as intermediates pH-dependence indicates 2 important His, 12, 119 that act in a concerted manner as general acid and base catalysts to catalyze a two-step reaction

X-ray structure of bovine pancreatic RNase S

UpcA substrate in active site

The RNase A mechanism

B) Covalent Catalysis Usually Requires a Nucleophile

Covalent Catalysis accelerates reaction rates through the transient formation of a catalyst-substrate covalent bond Usually, nucleophilic group on enzyme attacks an electrophilic group on the substrate = nucleophilic catalysis Example: decarboxylation of acetoacetate

Decarboxylation of acetoacetate

Three stages of Covalent Catalysis

1. Nucleophilic attack of enzyme on substrate 2. Withdrawal of electrons 3. Elimination of catalysts by reversion of step 1 (not shown above).

Nucleophilicity of a substance is related to its basicity:

Important aspect of covalent catalysis

The more stable the covalent bond formed, the less easily it can be decomposed in the final step of a reaction
Good covalent catalysis must be (i) highly nucleophile and (ii) form a good leaving group. These are imidazole and thiol groups, i.e. Lys, His and Cys, Asp, Ser, some coenzymes (thiamine pyrophosphate, pyridoxal phosphate)

C) Metal Ion Cofactors Act as Catalysts

1/3 of known enzymes require metal ions for catalysis Metalloenzymes contain tightly bound metal ion (Fe2+, Fe3+, Cu2+, Mn2+, Co2+), Na+, K+, or Ca2+ play structural rather than catalytic roles Mg2+, Zn2+ may be either structural or catalytic

Metal Ion Cofactors Act as Catalysts

Metal ions participate in the catalytic process: 1. By binding to substrate to orient them properly for reaction 2. By mediating oxidation-reduction reactions through reversible changes in the metal ions oxidation state 3. By electrostatically stabilizing or shielding negative charges Often: Metal ion acts similar to a proton, or polarizes water to generate OH-

The role of Zn2+ in carbonic anhydrase

CO2 + H2O <-> HCO3- + H+ Zn2+ polarizes water, which then attacks CO2

D) Catalysis can occur through proximity and orientation effects

Enzymes are much more efficient catalysts than organic model compounds Due to proximity and orientation effects Reactants come together with proper spatial relationship
Example: p-nitrophenylacetate intramolecular reaction is 24 times faster

Inter- versus intramolecular reaction

24-times faster

Catalysis can occur through proximity and orientation effects

Enzymes are usually much bigger than their substrates By oriented binding and immobilization of the substrate, enzymes facilitate catalysis by four ways 1. bring substrates close to catalytic residues 2. Binding of substrate in proper orientation (up to 102-fold) 3. Stabilization of transition state by electrostatic interactions 4. freezing out of translational and rotational mobility of the substrate (up to 107-fold)

The geometry of an SN2 reaction

E) Enzymes catalyze reactions by preferentially binding the transition state

An enzyme may binds the transition state of the reaction with greater affinity than its substrate or products This together with the previously discussed factors accounts for the high rate of catalysis For example, if enzyme binds the transition state with 34.2 kJ/mol (= 2 hydrogen bonds) it results in 106-fold rate enhancement

315-times faster if R is CH3 rather than H

Effect of preferential transition state binding

Transition state analogs are enzyme inhibitors

For example proline racemase


4) Lysozyme
Lysozyme is an enzyme that degrades bacterial cell walls.
Hydrolyzes (1->4) glycosidic bond from N-acetylmuramic (NAM) acid to N-acetylglucosamine (NAG) in cell wall peptidoglycan also hydrolyzes chitin: (1->4) NAG Lysozyme occurs widely as bactericidal agent, best characterized: hen egg white lysozyme, 14.3 kD, single 129 Aa polypeptide chain, 4 disulfide bonds, rate enhancement 108-fold

The lysozyme cleavage site


Lysozymes catalytic site was identified through model

Lysozyme structure solved by X-ray in 1965, first enzyme Ellipsoidal shape with prominent cleft in substrate bdg site, That traverse one face of the molecule Use model building to understand enzyme substrate interactions 6 saccharide units, A-F In D ring, C6 and O6 too closely contact enzyme => distortion of glucose ring from chair => half chair => have to move from

Lysozymes catalytic site was identified through model

Chair and half-chair conformation

Distortion of D ring, Saccharide unit 4 => C1, C2, C5 , and O5 are coplanar Stabilization through H bridges and ionic interactions

The interactions of lysozyme with its substrate

Identification of the bond that lysozyme cleaves

D-ring remains

B) The lysozyme reaction proceeds via a covalent intermediate

The reaction catalyzed by lysozyme, the hydrolysis of a glycoside, is the conversion of an acetal into a hemiacetal Non-enzymatic, this is an acid-catalyzed reaction, involving the protonation of an oxygen atom, followed by cleavage of a O-C bond -> transient formation of resonance stabilized carbocation = oxonium ion Enzymatic reaction should include an acid catalyst and a stabilization of the oxonium ion transition state

The mechanism of the nonenzymatic acidcatalyzed hydrolysis of an acetal to a hemiacetal

Glu 35 and Asp 52 are lysozymes catalytic residues

NAG lactone binds to the D subside with about 9.2 kJ/ mol greater affinity than does NAG (corresponds to a 40fold enhancement)

Transition state analog inhibition of lysozyme

Observation of the covalent intermediate

The lifetime of a glucosyl oxonium ion in water is ~10-12 sec To be observed: its rate of formation must be greater than that of its breakdown
1. Formation slowed by substituting F at C2 of D ring to draw electrons 2. Mutating Glu 35 to Gln (E35Q) to remove general acid base catalyst 3. Substitution F at C1 of D ring as good leaving group

4) Serine Proteases

Class of proteolytic enzymes,
Active site reactive Ser-residue (cut after Ser !) digestive enzymes, developmental regulation blood clotting inflammation many other cellular processes Focus on chymotrypsin, trypsin, elastase

A) Active site residues were identified by chemical labeling

Chymotrypsin, trypsin, elastase are digestive enzymes synthesized by the pancreas, secreted into duodenum All cleave peptide bonds but with different specificities for side chain residues Chymotrypsin: after bulky hydrophobic residue Trypsin: after positively charged residue Elastase: after small neutral residue Chemical labeling with diisopropylphosphofluoridate (DIPF) Reacts only with Ser 195 of chymotrypsin, very toxic Does not label other Ser, why ?

Diisopropylphosphofluoridate (DIPF)

Diisopropylphosphofluoridate (DIPF)

A second important residue, His 57, was identified by affinity labeling Substrate analog bearing reactive groups reacts with nearby residues, Trojan horses Chymotrypsin specifically binds tosyl-L-phenylalanine chloromethylketone (TPCK), resembles Phe, reacts with His 57

B) X-ray structures provide information bout catalysis, substrate specificity, and evolution
Chymotrypsin, trypsin, elastase are strikingly Similar Have ca. 240 Aa, 40% identical All have reactive Ser and important His Closely related 3D structure, chymotrypsin solved in 1967 Active site His 57, Ser 195, Asp 102 form Catalytic triad residues

X-ray structure of bovine trypsin in complex with leupeptin

The active site residues of chymotrypsin

Nerve Poisons

Use of DIPF as enzyme inhibitor based on discovery that organophosphorous compounds, such as DIPF, acts as potent nerve poisons. Inactivate acetylcholinesterase, catalyzes hydrolysis of acetylcholine, active site Ser

Nerve Poisons

Acetylcholine is a neurotransmitter: transmits nerve impulses across certain types of synapses (junctions between nerve cells) Acetylcholinesterase in the synaptic clevt normally degrades acetylcholine to terminate nerve impulse. Acetylcholine receptor, which is a Na+-K+ channel, remains open for longer than normal, toxic to humans (inability to breathe) DIPF so toxic that it has been used as military nerve gas. Related compound such as parathion and malathion are used as insecticides

Used by terrorists in Tokyo subway, 1995 Inactivated by paraoxonase, expressed at different levels in different individuals, different sensitivity to nerve toxins of this class

Tetrahedral phosphate = transition state analog

Substrate specificities are only partially rationalized

X-ray structure suggest the basis for the Different substrate specificities of chymostrypsin,trypsin and elastase 1. In chymotrypsin, preferred Phe, Trp or Tyr fit into a slitlike hydrophobic pocket located near the catalytic groups

Specificity pockets of three serine proteases

2. In trypsin, the Ser 198 of chymotrypsin, which lies at the bottom of the binding pocket is replaced by Asp. Form ion pairs with Arg and Lys in substrate. But equally deep slitlike pocket as in chymotrypsin
But Asp->ser 189 mutation does not convert Trypsin into chymotrypsin

Specificity pockets of three serine proteases

3. In elastase, hydolyzes the nearly indegstible Ala, Gly, and Val-rich protein elastin (connective tissue) Bdg pocket contains Val and Thr instead of the two Gly found in trypsin and chymotrypsin -> cleaves substrates with small neutral side chains

Serine proteases exhibit divergent and convergent evolution

Great overall similarity -> arose through duplication of an ancestral enzyme, followed by divergent evolution of the resulting enzyme Primordial enzyme arose before separation of pro- and eukaryote Other Ser-proteases, however, have very little homology, i.e, subtilisin and serine carboxypeptidase II Arose through convergent evolution

C) Serine proteases use several catalytic mechanisms

Catalytic mechanism of chymotrypsin, based on structural and chemical data. Applies to all Ser proteases and other hydrolytic enzymes (lipases.) 1. After chymotrypsin has bound substrate: Ser 195 nucleophilic attack on peptides carbonyl group to form tetrahedral intermediate, resembles transition state of this covalent catalysis, Proton on Ser is abosrbed by His 57 to fomr imidazolium ion (general base catalysis), aided by Asp 102

Formation of the tetrahedral intermediate

2. Decomposition of the tetrahedral intermediate

Decomposition to the acylenzyme intermediate and scission of the peptide bond Driven by donation of proton from N3 of His 57 (general acid catalysis) Helped by polarizing effect of Asp 102 on His 57 (electrostatic catalysis)

3. Amine leaving group is replaced by water

The amine leaving group (the new N-terminus of the cleaved peptide) is released from the enzyme and replaced by water from the solvent

4. Hydrolysis of the acylenzyme intermediate

By the addition of water, formation of a second tetrahedral intermediate

5. Reversal of step 1

Yields the carboxylate product, that is the new Cterminus of the peptide, and regenerates the active enzyme

Serine proteases preferentially bind the transition state

1. Conformational distortion that occurs with formation of the tetrahedral intermediate causes the anionic carbonyl oxygen to move deeper into the active site so as to occupy the oxyanion hole 2. There it forms two hydrogen bonds with the enzyme the oxyanion hole is conserved in chymotrypsin and subtilisin, convergent evolution 3. This tetrahedral distortion allows formation of another hydrogen bond between Gly 193 and the backbone NH of the residue preceding the scissile peptide bond

Transition state stabilization in the serine proteases

Transition state stabilization in the serine proteases

The preferential binding of the transition state (or the tetrahedral intermediate) over the enzyme-substrate complex or the acyl-enzyme intermediate is responsible for much of the catalytic efficiency of serine proteases
Mutating any or all residues of the catalytic triad yields enzymes that still enhance proteolysis by ca. 5 104-fold over the noncatalyzed reaction, native enzyme 1010

Low-barrier hydrogen bonds may stabilize the transition state

1. Proton transfer between hydrogen donor and acceptor occurs at reasonable rates only when the pK of the donor is 2-3 pH units greater than that of the protonated form of the acceptor 2. If their pK values of proton donor and acceptor are nearly equal, the distinction breaks down and: the hydrogen atom becomes more or less equally shared between them (D---H---A). 3. Such low-barrier hydrogen bonds (LBHBs) are unusually strong and short (40-80 kJ/mol versus 12-30 kJ/mol; 2.55-2.65 versus 2.8-3.1) 4. LBHBs dont exist in aqueous phase but can form in the environment of an enzyme

The tetrahedral intermediate resembles the complex of trypsin with trypsin inhibitor

1. Strong evidence for formation of a tetrahedral intermediated provided by X-ray structure of trypsin with bovine pancreatic trypsin inhibitor (BPTI) BPTI, 58 Aa, prevents self-digestion of organ of prematurely activated trypsin, k= 1013 Mol, one of the strongest protein interactions known A Lys on BPTI occupies trypsins specificity pocket But proteolytic reaction cannot proceed because the active site is so tightly sealed that the leaving group does not dissociate and water cannot enter Protease inhibitors are common, e.g. plant defence against insects, 10% of blood plasma (a1-proteinase inhibitor against leukocyte elastate (inflammation))


3. 4.


The tetrahedral intermediate resembles the complex of trypsin with trypsin inhibitor

The tetrahedral intermediate has been directly observed

Since the tetrahedral intermediate resembles the transition state, it is thought to be unstable and short-lived. Acly-enzyme complex is table at pH 5.0 (His 57 is protonated an cannot act as base catalyst) and could be observed by X-ray Immersing the acyl-enzyme crystals a pH 9 triggers the hydrolytic reaction Freeze crystals in liquid N2 and analyze by X-ray

Structure of the acyl-enzyme and tetrahedral intermediates

D) Zymogens are inactive enzyme precursors

Proteolytic enzymes are usually made as larger, inactive precursors = zymogens (proenzymes) Acute pancreatitis is characterized by premature activation of digestive enzymes Enteropeptidase converts trypsinogen into trypsin, Ser-protease under hormonal control, made in the duodenal mucosa, cleaves lys 15 Ile 16 = trypsin cleavage site, i.e. self activation / autocatalytic Also proelastase, procarboxypeptidase A, B, and prophospholipase A2 are all activated by trypsin

The activation of trypsinogen to trypsin

Zymogens have distorted active sites

Liberation of N-terminal peptide results in conformational change and activation of the enzyme

The blood coagulation cascade

If blood vessel is damaged, clot forms as result of platelet aggregation (small enucleated blood cells) and formation of insoluble fibrin network that traps additional blood cells Fibrin is produced from the soluble circulating fibrinogen through activation of the ser protease thrombin Thrombin is the last enzyme in a coagulation cascade of enzymes, activation occurs on platelets Initiated by membrane protein, tissue factor, forms complex with circulating factor VII (extrinsic pathway)

The blood coagulation cascade

The blood coagulation cascade

Intrinsic pathway activated by glass surface (negative charge) Congenital defects in factor VIII (hemophilia a) or factor IX (hemophilia b)