ENZYME MECHANISMS

Today we will consider aspects of the spectacular ability of enzymes to catalyze chemical reactions. We will cover:

1) General Aspects. Enzymes catalyze reactions by lowering the activation

energy barrier for the reaction and often providing alternate pathways for a reaction to occur. We will discuss four general categories of catalytic mechanisms including a) transition state stabilization, b) general acid-base catalysis, c) covalent catalysis and d) metal ion catalysis.

2) Classifications of Enzymes. Six different general classes of enzymes will be

discussed in order to provide a conceptual framework for enzymes that will be covered in the course.

3) Proteases. As examples of enzymes with important medical consequences,

we will discuss the properties and mechanisms of proteases, including four major types of proteases. We will finish with a discussion of the mechanistic aspects of catalysis by serine proteases.

Reading: Lippincott Chapter 5, sections I - IV

I. General Aspects of enzymatic reactions and transition states

Consider a reaction A + B P + Q
A +B K

k'

P +Q

where A + B react through transition state, X, to form products P + Q. K is the equilibrium constant between A + B and X and k' is the rate constant for conversion of X to P + Q.
G G A +B G reaction P +Q Reaction coordinate The minimum energy pathw ay of the reaction is show n in the reaction coordinate, or trans ition state diagram, at left. Chemical conversion ofA + B to P + Q proc eeds through a transition state which is the leas t stable (leas t probable, highest free energy) spec ies along the pathw ay. Molecules that ac hieve the activation energy, G , can go on to react w hile molecules that fail to achieve the transition state fall bac k to the ground state.

The transition state, X, is metastable with only a transient existence. The less stable the transition state, the more difficult it is for a reaction to proceed.

The smaller the difference in free energy of the reactants and the transition state, the faster the reaction proceeds. Enzymatic rate accelerations are achieved by lowering the activation barrier between reactants and the transition state, thereby increasing the fraction of reactants able to achieve the transition state. Enzymes catalyze reactions by either stabilizing the normal transition state or providing an alternative pathway from reactants to products.

a) Transition state stabilization by induced fit b) Acid-base catalysis

c) Covalent Catalysis
d) Metal ion catalysis

a) Transition state stabilization

Imaginary enzyme ("stickase") designed to catalyze "cleavage" (breaking) of a metal stick ("magnetic" interactions, red dashed lines, represent noncovalent interactions between enzyme and substrate and between enzyme and transition state ). Metal stick must be bent, a "high energy state", before it can be broken, so "transition state" is bent stick.
(Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000)

No Enzyme

No catalysis is obtained by just binding substrate tightly!

b) General acid-base catalysis:

Often used in the hydrolysis of ester/ peptide bonds, phosphate group reactions, addition to carbonyl groups, etc. An enzyme avoids unstable charged intermediates in reaction (which would have high free energies) by having groups appropriately located to: donate a proton (act as a general acid), or

What side-chains can act donate or accept protons?

Amino Acid
Aspartic acid

pK a
3.90

COO H C CH
+ NH 3 2

-COOH O C O-COOH O CH 2 C Oimidazole N N sulf hydryl SH phenol

2

COO Glutamic acid 4.07 H C CH 2 + NH 3 COO Histidine 6.04 H C CH 2 + NH 3 COO Cysteine 8.33 H C CH 2 + NH 3 COO Tyrosine 10.13 H C CH
+ NH 3

OH -amino CH 2 CH 2 NH + 3

accept a proton (abstract a proton, act as a general base)

COO Lysine 10.79 H C CH 2 + NH 3

c) Covalent catalysis (also sometimes called nucleophilic catalysis):

(transient formation of a catalyst-substrate covalent bond)

Biologically important nucleophilic groups:

Nucleophilic form

Hydroxyl group Sulfhydryl group Amino group Imidazole group

R-OH R-SH
+ R-NH3

R-O:
R-S: R-NH2
R
HN N:

+ H+ + H+ + H+ + H+

R
HN + NH

Biologically important electrophiles:

H+
Protons

Mn+
Metal Ions

C=O
Carbonyl carbon

R-NH2

+ C=O

Adapted from Voet & Voet, Biochemistry

d) Metal ion catalysis:

Metal ions are often used for one or more of the following: * binding substrates in the proper orientation * mediating oxidation-reduction reactions * electrostatically stabilizing or shielding negative charges (electrostatic catalysis)

Metalloenzymes contain tightly bound metal ions: (usually Fe+2, Fe+3, Cu+2, Zn+2, or Mn+2)
Metal-activated enzymes contain loosely bound metal ions: (usually Na+, K+, Mg+2, or Ca+2)

Which of the following mechanisms is not used by enzymes for catalysis? A. Acid base catalysis B. Induced fit of intermediate C. Providing complementary electrostatics D. Binding of metal ions E. Destabilizing the transition state Which of the following residue side-chains could not contribute to acid-base catalysis? A. Lysine

B. Glutamate
C. Histidine D. Phenylalanine

E. Tyrosine

II. Enzyme Classes

Enzymes have been grouped into 6 general classes by The Enzyme Commission. These include 1) Oxidoreductases, 2) Transferases, 3) Hydrolases, 4) Lyases, 5) Isomerases and 6) Ligases. Since such classifications can aid recognition of enzymes, we will briefly discuss each of these groups and provide examples.

1) Oxidoreductases: This is a very broad class of enzymes that catalyze the many
oxidation-reduction reactions found in biochemical pathways. Oxidoreductases catalyze reactions in which at least one substrate gains electrons, becoming reduced, and another loses electrons, becoming oxidized.

An important subset of oxidoreductases are the dehydrogenases that accept and donate electrons as hydride ions (H:-) or hydrogen atoms often using cofactors such as NAD+/NADH as an electron donor or acceptor. An example is Lactate Dehydrogenase (LDH) (right).

LDH

NAD+

NADH

(From Marks Basic Medical Biochemistry A clinical approach)

2) Transferases: This class of enzymes catalyze

the transfer of a specific functional group between molecules. Important subsets of transferases include a) Kinases that transfer phosphate groups, usually from ATP to another molecule (such as hexokinase and glucokinase, that phosphorylate glucose and protein kinases that phosphorylate protein hydroyxl groups), b) Aminotransferases (see right) that transfer amino groups that are important in amino acid metabolism, c) Acyltransferases that transfer fatty acyl groups and d) Glycosyltransferases, which transfer carbohydrate residues.
Aminotransferase reaction in which Pyridoxal phosphate (PLP) is used as a cofactor. (From Marks Basic Medical Biochemistry A clinical approach)

3) Hydrolases:

Hydrolysis reactions refer to the cleavage of bonds by the addition of a water molecule. A very important class of hydrolases are the proteases involved in cleaving peptide bonds, as we will be discussing shortly

4) Lyases: This class refers to those enzymes involved in cleaving bonds by means other than hydrolysis or oxidation. Examples include aldolases (such as fructose diphosphate aldolase, which is involved in glycolysis) and thiolases (such as ketoacyl-CoA thiolase involved in the breakdown of fatty acids). Lyases also include enzymes involved in elimination of groups from two adjacent carbon atoms to form double bonds.
In glycolysis, fructose 1,6bisphosphate aldolase cleaves a carbon-carbon bond in fructose 1,6-bisphosphate. (From Marks Basic Medical Biochemistry A clinical approach)

5) Isomerases: At many stages of metabolism,

rearrangement of the atoms of a molecule is required to create an isomer of the starting compound. Enzymes generally catalyzing the rearrangement of the bond structure are called isomerases, while those specifically catalyzing the movement of a phosphate from one group to another are known as mutases. For example, triose phosphate isomerase (right) catalyzes the interconversion between dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate, which is essential for continuing glycolysis following splitting of six carbon sugars into two three carbon sugars by fructose diphosphate aldolase

(From Marks Basic Medical Biochemistry A clinical approach)

6) Ligases: Ligases are involved in synthesizing bonds between carbon atoms and carbon, nitrogen, oxygen or sulfur atoms in reactions that are coupled to the cleavage of the high energy phosphate of ATP or another nucleotide. Pyruvate carboxylase, a key enzyme in gluconeogenesis, is one important ligase in metabolism.

Proteases cleave peptide bonds by the addition of water. To what class of enzymes do they belong? A. Oxidoreductases B. Ligases C. Hydrolases D. Lyases E. Isomerases

15

III. Proteases - Enzymes that specifically cut other proteins and are important in regulation

(Ser, Cys or Thr) (Asp or Glu) (usually His)

(metal)

Protease classification 1) Cysteine Proteases

2) Aspartyl Proteases
3) Metalloproteases 4) Serine Proteases

1) Cysteine Proteases
MECHANISM: In cysteine proteinases, catalysis proceeds through the formation of a covalent intermediate and involves a cysteine and a histidine residue. The essential Cys and His play the same role as Ser and His respectively in Serine proteases as discussed later. The nucleophile is a thiolate ion that is stabilized through the formation of an ion pair with neighbouring imidazolium group of His. The attacking nucleophile is the thiolate-imidazolium ion pair in both steps. EXAMPLES: Medically interesting cysteine proteases include: - mammalian enzymes such as cathepsins B and L, which are involved in cancer growth and metastasis, and cathepsin K, which is important for bone degradation an osteoporosis. - Cruzipain and cruzain from Trypanosoma cruzi, which cause Chagas' disease, a permanent infection that affects more than 25 million people annually in South America and causes more than 45,000 deaths per year, and falcipain, from Plasmodium falciparum, which causes malaria. - Caspases, which are key mediators of apoptosis.

2) Aspartyl Proteases
MECHANISM: In contrast to cysteine (and serine) proteases, catalysis by aspartic proteinases do not involve a covalent intermediate, even though a tetrahedral intermediate is transiently formed. Rather, nucleophilic attack is achieved by two simultaneous proton transfers: one from a water molecule to one of the two carboxyl groups and a second one from the carbonyl oxygen of the substrate with the concurrent CO-NH bond cleavage. This general acid-base catalysis, which may be called a "push-pull" mechanism leads to the formation of a non covalent neutral tetrahedral intermediate EXAMPLES: Plasmepsin, which is produced in the parasite that causes malaria, is part of a closely related group of enzymes known as aspartyl proteases. Plasmepsin is believed to play a key role in the digestion of the human host's hemoglobin, the major nutrient source for the parasite. HIV protease permits viral maturation. BACE, an aspartyl protease involved in the amyloid peptide generation of Alzheimer's disease.

3) Metalloproteases (Zn)
MECHANISM: Many enzymes contain the sequence HEXXH, which provides two histidine ligands for binding of zinc. A third Zn ligand is either a glutamic acid (thermolysin, neprilysin, alanyl aminopeptidase) or a histidine (astacin, serralysin). Other families exhibit a distinct mode of binding of a Zn ion. The catalytic mechanism involves formation of a non covalent tetrahedral intermediate after the attack of a zinc-bound water molecule on the carbonyl group of the scissile bond. This intermediate can be further decomposed by transfer of the glutamic acid proton to the leaving group. EXAMPLES: Matrix metalloproteinases (MMPs) are a family of enzymes that are responsible for the degradation of extracellular matrix components such as collagen, laminin and proteoglycans. These enzymes are involved in normal physiological processes such as embryogenesis and tissue remodeling and may play an important role in arthritis, periodontitis, and metastasis. ACE is a metalloprotease that catalyses the conversion of angiotensin I into angiotensin II, which leads to vasoconstriction. ACE inhibitors, were originally used as antihypertensives, but have significantly improved the treatment of other cardiovascular diseases and are now used to treat heart failure and even prevent heart attacks in at-risk patients.

4) Serine Proteases
MECHANISM: The key active site groups are Ser, His and Asp. These groups are in the same orientation in all the serine proteases. Their roles are basically as follows: the imidazole (His) acts as a general base-general acid, first to activate the serine OH for nucleophilic catalysis, then the leaving group (by general acid cat.), then as a general base it activates water to attack the covalent acyl-enzyme intermediate. The Asp serves to orient the His side chain and to provide an appropriate electrostatic environment. EXAMPLES: Trypsin and Chymotrypsin are digestive enzymes in the small intestine Subtilisin is a bacterial serine protease that is used in laundry detergents TADG-14 is a novel extracellular serine protease that has been identified and cloned from ovarian carcinoma. It is uniquely expressed in ovarian cancer, both in early stage and overt carcinomas. It is seldom or not at all expressed in normal adult tissues and has not been detected in other fetal tissues. It offers the potential as a target for therapeutic intervention through down-regulation of its protease activity. NS3/4A is a serine protease in Hepatitis C (HCV) that is important in viral maturation. Factor VIIa, Factor Xa, and thrombin are serine proteases in blood coagulation pathway

Trypsin and Chymotrypsin are very well studied serine proteases whose structures and mechanisms are well understood They catalyze the hydrolysis of internal peptide bonds (thus an endoprotease). Trypsin cleaves on the carboxyl side of basic side chains (Lys, Arg), whereas chymotrypsin cleaves on the carboxyl side of aromatics (Phe, Tyr, Trp) The active site consists of a catalytic triad: 1) Serine, to which the substrate binds 2) Histidine, which has the ability to donate and accept protons. 3) Aspartate, which has the ability to accept protons. These residues are polar (hydrophilic) so would not ordinarily be found on the "interior of a protein. Though they are in close proximity in the 3D structure, they are not adjacent in the primary sequence (Ser-195, His-57, Asp-102).
102

First stage in peptide bond hydrolysis: acylation. Hydrolysis of the peptide bond starts with an attack by the oxygen atom of the Ser195 hydroxyl group on the carbonyl carbon atom of the susceptible bond. The carbon-oxygen bond of this carbonyl group becomes a single bond, and the oxygen atom acquires a net negative charge. The four atoms now bonded to the carbonyl carbon are arranged as a tetrahedron. Transfer of a proton from Ser195 to His57 is facilitated by Asp102 which (i) precisely orients the imidazole ring of His57 and (ii) partly neutralizes the positive charge that develops on His57 during the transition state. The proton held by the protonated form of His57 is then donated to the nitrogen atom of the peptide bond that is cleaved. At this stage, the amine component is hydrogen bonded to His57, and the acid component of the substrate is esterified to Ser195. The amine component diffuses away.
Oxyanion hole

Second stage in peptide hydrolysis: deacylation. The acyl-enzyme intermediate is hydrolyzed by water. Deacylation is essentially the reverse of acylation with water playing the role as the attacking nucleophile, similar to Ser195 in the first step. First, a proton is drawn away from water. The resulting OH- attacks the carbonyl carbon of the acyl group that is attached to Ser195. As in acylation, a transient tetrahedral intermediate is formed. His57 then donates a proton to the oxygen atom of Ser195, which then releases the acid component of the substrate, completing the reaction.

Oxyanion hole

(From Marks Basic Medical Biochemistry A clinical approach)

Activation Strategies for three classes of Proteases

From: Berg, Tymoczko and Stryer: Biochemistry, 5th edition

How is specificity obtained among trypsin-like serine proteases?

Are all serine proteases evolutionarily related? (NO!)

Catalytic triad: Ser 195, His 57, Asp 102

Catalytic triad: Ser 221, His 64, Asp 32

Which of the following peptides would trypsin cleave? A. Asp-Leu-Trp-Ala-Leu-Met-Tyr B. Asn-Thr-Ser-Asp-Ala-Leu-Gly C. His-Ala-Asp-Val-Asn-Gln-Ala D. Val-Ala-Val-Lys-Ser-Gly-Phe E. Phe-Ala-Trp-Ser-Ile-Ala-Gly

The physiological importance of many proteases has made them an extremely active objects of current research:

Highly selective small organic compounds are being investigated as therapeutic leads against many proteases.
This is one of the hottest areas of drug development in the biotech and pharmaceutical industry Many compounds are in preclinical or clinical trials.

Many of these compounds will likely be drugs by the time you are doctors!

Summary Points on Enzyme Catalysis:

1) Understand what is meant by transition state stabilization. 2) Know which amino acid residues can be involved in acid-base catalysis. 3) Understand that covalent catalysis results from the transient formation of a covalent bond involving a nucleophile and an electrophile. 4) Recognize the general classes of enzymes, including oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. 5) Recognize the physiological importance of Proteases.

6) Understand that proteases use nucleophilic attack by either covalent catalysis or activation of a water molecule, along with acid-base catalysis.
7) Know what is meant by a serine protease. 8) Have a general understanding of covalent catalysis by Trypsin. 9) Understand that the specificity pocket allows different members of the trypsin family of proteases to use the same catalytic mechanism for proteolysis but with very different sequence specificities.