**ENZYMES**

Definitions-Chemical reactions in cells require specific catalysis. Enzymes are proteins which perform this function. Metabolite acted upon is called the enzymes substrate.

A. Fundamental Properties 1) Enzymes are excellent catalysts, speeding up reactions 108 to 1020 fold. They speed up reactions without being used up. 2) Specificity a) for substrate - ranges from absolute (e.g., aspartase) to relative b) for reaction catalyzed, i.e.,few side-reactions and by-products, etc.) 3) Regulated-- some enzymes can sense metabolic signals.

B. Enzymes as Molecules 1) Large molecules-- proteins from 12kDa - 1,000kDa or more -- most are much larger than their substrate. 2) Active site -- specific region in enzyme which interacts with its substrate.

both binding and catalytic reaction occur here. some residues involved in binding substrate others catalyze reaction

3) Cofactors for some reactions, the amino acids are not powerful enough for catalysis. some enzymes incorporate additional factors. metal ions as cofactors-- Zn2+, Fe2+, Cu2+, others coenzymes are organic cofactors prosthetic groups are covalently attached

C. Classification of Enzymes 1) named and classified according to the substrate acted upon and the reaction catalyzed. 2) trivial names-- end in -ase -- urease, hexokinase. 3) named based on a formal systemic catalog (IUB) with six major classifications. (All enzymes should fall into one of these categories and all enzymes therefore have a formal name.)

Class 1. Oxidoreductases- catalyze redox processes Example: RCH2-OH RCH=O

Class 2. Transferases- transfer chemical groups from one molecule to another or to another part of the same molecule. O O Example: CH3-C-SCoA + XR CH3-C-XR + HSCoA acetyl CoA acetyl group transferred

Class 3. Hydrolases- cleave a bond using water to produce two molecules from one. O H 2O O example: --CNH-R --C-OH + H2N-R cleavage of a peptide bond Class 4. Lyases- remove a group from or add a group to double bonds. H-X H X ---C=C--- ---C--C---

Class 5. Isomerases- interconvert isomeric structures by molecular rearrangements. CH3 CH3 HC-OH HO-CH COOH COOH Class 6. Ligases -- join two separate molecules by the formation of a new chemical bond usually with energy supplied by the cleavage of an ATP. example: O ATP ADP+Pi O -OOC-C-CH + CO -OOC-C-CH -COO3 2 2 pyruvate oxaloacetate enzyme = pyruvate carboxylase

Enzyme Mechanism Enzymes catalyze difficult reactions by changing the reaction to a series of easier steps including nucleophilic attack, general acid-base catalysis, covalent attachment, etc. Most common detailed example is action of chymotrypsin, a protease.

Coenzymes and Vitamins Some coenzymes are loosely held or transiently bound acting more or less as second substrates & others are tightly held within the protein. The latter are called prosthetic groups. Water-Soluble Vitamins

Coenzyme = thiamine pyrophosphate (TPP) use = decarboxylation and transketolation its vitamin = thiamine or vitamin B1, contains pyrimidine and thiazole. disease = beri-beri, Wernikes disease. peripheral nerves, muscle cramps, numbness

Coenzyme = flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) both act as prosthetic groups -- use = redox reactions -- its vitamin = riboflavin or B2

riboflavin

nicotinamide-adenine dinucleotide (NAD), nicotinamide-adenine dinucleotide phosphate (NADP) Coenzyme -- use = redox reactions with H transfer -- its vitamin = P niacin or B3 = nicotinamide & nicotinic acid -- disease = pellagra, skin lesions, swollen tongue, nervous/mental disorders

Coenzyme = pyrodoxal phosphate -- use = decarboxylations, transaminations and racemases -- its vitamin = pyridoxine, or vitamin B6

Coenzyme = Coenzyme A (CoA) -- use = activates carbonyl groups and in acyl transfer (acetyl- CoA, synthesis of fats and steroids) -- its vitamin = pantothenic acid -- disease= GI problems, emotional instability, burning sensation in extemities

acetyl Acetyl CoA

Coenzyme = folate or tetrahydrofolate (the reduced form) -- use = transfer of one carbon unit or formate -- its vitamin = folic acid -- disease = megablastic anemia, birth defects

Coenzyme = biotin a prosthetic group -- use = carboxylations -- its vitamin = biotin

Coenzyme = cyanocobalamin -- use = methyl group transfer; folate metabolism, myelin synthesis -- its vitamin = cyanocobalamin or vitamin B12 -- disease = pernicious anemia

Coenzyme = lipoic acid (reduced SH or oxidized form -S-S-) prosthetic group (recall oxidized pyruvate dehydrogenase) -- use = redox reactions -- its vitamin = lipoic acid (humans probably produce enough so it is not always considered a vitamin) reduced

ascorbic acid or vitamin C -- Not a coenzyme, a cosubstrate -- use = antioxidant (aqueous phase), Hydroxylations (collagen)
-- disease = subacute scurvy, sore and bleeding gums, loose teeth, fatigue, lack of resistance to disease. Scurvy itself is very unusual.

Fat-Soluble Vitamins
The following fat-soluble vitamins are also generally not coenzymes but are included here for completion.

Vitamin A -- its derivative retinal is the chromophore or light absorbing factor in rhodopsin, our visual protein -- use = vision, bone formation,differentiation of epithelial cells, early development -- disease = night blindness, sterility, skin lesions

Vitamin D -- use = calcium and phosphate absorption and metabolism -- disease = rickets in children, osteomalacia in adults

Vitamin E, tocopherol -- use = antioxidant (lipid phase), free radical trapping -- disease = liver atrophy, hemolysis of erythrocytes

Vitamin K fat soluble -- use = blood coagulation (needed for synthesis of prothrombin), biosynthesis of Ca binding proteins -- disease = slow clotting time, excessive bleeding

Reaction Rates and the Transition State Enzymes speed up reactions enormously. To understand how they do this, examine the concepts of activation energy & the transition state. In order to react, the molecules involved are distorted, strained or forced to have an unlikely electronic arrangement. That is the molecules must pass through a high energy state.

This high energy state is called the transition state. The energy required to achieve it is called the activation energy for the reaction.

The higher the free energy change for the transition barrier, the slower the reaction rate.

Enzymes lower energy barrier by forcing the reacting molecules through a different transition state. This transition state involves interactions with the enzyme.

Enzyme

Modes of Enzymatic Enhancement of Rates 1) general acid and general base catalysis-- good proton donors & acceptors positioned just right. 2) covalent catalysis- unstable intermediate 3) metal ion catalysis - electron donor or acceptor 4) electronic effects- orbital steering of Koshland, steering aromatic groups

5) proximity and orientation - close to reactive group and aligned versus random in solution chemistry.

6) conformational strain distortion and induced fit old idea, lock-and-key= substrate fits active site

induced fit- enzyme changes its conformation to accept the transition state of substrate/product well. Enzyme conformational change works to distort and strain substrate forcing it into transition state. Simultaneous Koshland

ENZYME KINETICS Why study enzyme kinetics? a) the precise scheduling of reactions in a cell is important to the cell and our understanding of its workings b) enzyme mechanisms, e.g., the number of kinetic steps and the detailed chemistry can be learned (enzymology). c) understanding enzyme function leads to better drugs.

Definition: rate of a reaction For an enzyme acting on its substrate, just as an ordinary chemical reaction, the rate of the reaction depends on the concentration of substrate, S. A reaction leading to formation of product is written: S P Rate = change in [P]/ change in time or rate = v = [P]/t

For a chemical reaction (as contrasted to an enzymatically catalyzed one), the rate is proportional to reactant [S].

A rate constant, k, can be defined: rate

rate = v = [P]/t = k [S]

[S]

- In contrast, found empirically for enzymes: rate depends on [S] but hyperbolic curve & plateaus rate

rate also depends on the enzyme concentration [S]

Michaelis-Menten Model or Interpretation E + S ES E + P

where ES is enzyme-substrate complex, i.e., an intermediate complex. rate stops increasing or plateaus because the complex ES becomes filled at high [S]

Assigning rate constants to MM model: k1 k3 E + S ES E+ P k2 From this kinetic scheme, a relationship can be derived for the rate or velocity of the reaction: Michaelis-Menten Equation Vmax[S] V= [S] + Km gives hyperbolic curve on next slide.

Vmax, the maximum rate (plateau) is k3 x [total enzyme] Km =(k2 +k3)/k1, almost a binding constant

Vmax
Vmax is approached asymptotically Vmax/2

v or rate 0
0

Km [S]

Unit of velocity is moles/minmg protein

Km = [S], where the velocity v = Vmax /2, is called the Michaelis constant. Km is in units of concentration Km good estimate for the optimum Vmax concentration of substrate. Vmax/2 v Km [S]

A plot of v = Vmax[S] [S] + Km is not accurate enough to derive good Km & Vmax. Computer analysis is done. Reciprocal Plot A double reciprocal plot or Lineweaver-Burk plot is linear and more eye-appealing for presentation. mathematically = linear transformation

Result is:

1/v = Km/Vmax1/[S] + 1/Vmax

Looks like the linear y = mx + b m = slope b = intercept on y

1/v = Km/Vmax1/[S] + 1/Vmax x

Notice also intercept on x is -1/Km

1/v
intercept = 1/Vmax slope = Km/Vmax

-1/Km

1/[S]

Competitive Inhibition substrate presented as double reciprocal plot Model: E + S ES E + P + I inhibitor EI

I resembles S I binds at active site reversibly EI cannot bind to S so no reaction

Competitive Inhibition
Vmax No I +I

+more I

Km

In competitive inhibition, can always add enough [S] to overcome inhibition. same Vmax

Competitive inhibition
Double reciprocal plot

1/v

+ more I

Same 1/v intercept, same Vmax

+I

1/Vmax

Different slopes, competitive Inhibition changes apparent Km

No I

1/[S]

Note: inhibition line always above no inhibition.

Molecular interpretation for competitive inhibition competitive inhibitor binds to same site as the substrate (competes). its structure usually resembles substrate or product. While the inhibitor is bound, the enzyme cannot bind substrate and no reaction possible. Many pharmaceutical agents are competitive inhibitors so are many toxic substances.

example: captopril Blood pressure is regulated in kidney by renin, a specific proteolytic enzyme, which acts on angiotensinogen, the precursor for the active regulator. renin angiotensinogen angiotensin I asp-arg-val-tyr-ile-his-pro-phe-his-leu converting enzyme angiotensin II the active factor O peptide captopril HS-CH2-CH-C-N COOH captopril is ACE inhibitor CH3 pro-like here
(angiotensin converting enzyme}

Finding useful inhibitors Trial & error Molecular modeling Testing enzyme inhibition Testing safety Example: HIV Protease is a dimer. inhibitor is shown at active site. interactions involve both subunits.

Noncompetitive Inhibition substrate E + S ES E + P + + I I EI ESl inhibitor

EI and ESI not productive, depletes E and ES

Noncompetitive Inhibition 1/v

+I No I

1/[S]

different slopes, different 1/v intercepts.

Molecular Interpretation: Inhibitor binds the enzyme somewhere different from where the substrate binds. So the inhibitor does not care whether substrate is bound or not. Inhibitor changes the conformation of the enzyme at the active site so no reaction is possible with inhibitor bound.

EI and ESI not productive

Irreversible Inhibition reactive compounds

combine covalently to enzyme so as to permanently inactivate it (previous examples are all reversible)
almost all are very toxic most bind to a functional group in active site of enzyme to block that site

Example 1: diisopropyl fluorophosphate (DFP) binds covalently to serine in serine proteases & acetylcholinesterase - tool for biochemists sarin is a deadly nerve gas Paralysis O Isopropyl-O-P-O-CH2- AChE CH3

Example 2: penicillin and related antibiotics bind covalently to a peptidase involved in cell wall synthesis in bacteria
Staphylococci, Streptococci sp. and others