ENZYMES KINETICS, INHIBITION, REGULATION

Muhammad Jawad Hassan

Assistant Professor Biochemistry

V 0 varies with [S]

Michaelis-Menten kinetics V max approached asymptotically V 0 is moles of product formed per sec. when [P] is low (close to zero time) E + S  ES  E + P Michaelis-Menten Model V 0 = V max x[S]/([S] + K m ) Michaelis-Menten Equation

Steady-state & pre-steady-state conditions At equilibrium, no net change of [S] & [P] or of [ES] & [E] At pre-steady-state, [P] is low (close to zero time), hence, V 0 for initial reaction velocity

At pre-steady state, we can ignore the back reactions

Michaelis-Menten kinetics ( summary ) Enzyme kinetics (Michaelis-Menten Graph) : At fixed concentration of enzyme, to [S] when [S] is small, V 0 is almost linearly proportional but is nearly independent of [S] when [S] is large Proposed Model: E + S k  ES k  E + P ES complex is a necessary intermediate Objective: find an expression that relates rate of catalysis to the concentrations of S & E, and the rates of individual steps

Michaelis-Menten kinetics ( summary ) Start with: V 0 = k 2 [ES], and derive, V 0 = V max x[S]/([S] + K m ) At low [S] ([S] < K m ), V 0 = (V max /K m )[S] At high [S] ([S] > K m ), V 0 = V max When [S] = K m , V 0 = V max /2.

Thus, K

m

= substrate concentration at which the reaction rate (V

0

) is half max.

Range of K m values

K m provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Allosteric enzyme kinetics Sigmoidal dependence of V 0 on [S], not Michaelis-Menten Enzymes have multiple subunits and multiple active sites Substrate binding may be cooperative

Enzyme inhibition

A competitive inhibitor

Methotrexate A competitive inhibitor of dihydrofolate reductase role in purine & pyrimidine biosynthesis Used to treat cancer

K i = dissociation constant for inhibitor Kinetics of competitive inhibitor Increase [S] to overcome inhibition V max K m attainable, is increased

V max unaltered, K m increased Competitive inhibitor

Kinetics of non-competitive inhibitor Increasing [S] cannot overcome inhibition Less E available, V max K m is lower, remains the same for available E

Noncompetitive inhibitor K m unaltered, V max decreased

Enzyme inhibition by DIPF Group - specific reagents react with R groups of amino acids diisopropylphosphofluoridate DIPF (nerve gas) reacts with Ser in acetylcholinesterase

Affinity inhibitor: covalent modification

Catalytic strategies commonly employed

1. Covalent catalysis.

reactive group, The active site contains a usually a nucleophile that becomes temporarily covalently modified in the course of catalysis 2.

General acid-base catalysis.

A chemical reaction is catalyzed by an acid or a base. The acid is often the proton and the base is often a hydroxyl ion.

A molecule other than H 2 O may play the role of a proton donor or acceptor.

3.

Metal ion catalysis.

Metal ion can function in several ways; • can serve as an electrophile, stabilizing a negative charge on a reaction intermediate.

• can generate a nucleophile by increasing the acidity of a nearby molecule, such as H 2 O in the hydration of CO 2 by carbonic anhydrase.

• can bind to substrate, increasing the number of interactions with the enzyme.

4.

Catalysis by approximation.

Bringing two substrates together along a single binding surface on an enzyme

Enzyme specificity: chymotrypsin Cleaves proteins on carboxyl side of aromatic, or large hydrophobic amino acid Bonds cleaved, indicated in red The enzyme needs to generate a powerful nucleophile to cleave the bond

A highly reactive serine (#195) in chymotrypsin 27 other serines not reactive to DIPF, Ser 195 is a powerful nucleophile DIPF: di-isopropylphosphofluoridate, only reacts with Ser 195

Hydrolysis in two stages Covalent catalysis Acylation to form acyl-enzyme intermediate Ser 195 OH group attacks the carbonyl group Deacylation to regenerate free enzyme Acyl-enzyme intermediate is hydrolysed

Chymotrypsin in 3D 3 chains; orange, blue, & green Catalytic triad of residues, including Ser 195 2 interstrand, & 2 intrastrand disulfide bonds See Structural Insights Synthesized as chymotrypsinogen Proteolytic cleavage to 3 chains

The catalytic triad ( constellation of residues ) Ser 195 converted into a potent nucleophile , an alkoxide ion Asp 102 orients His 57 Imidazole N as base catalyst, accepts H ion, positions & polarizes Ser H ion withdrawal from Ser 195 generates alkoxide ion

Regulatory Strategies: Enzymes & Hemoglobin 1.

Allosteric control.

Proteins contain distinct regulatory sites and multiple functional sites.

Binding of regulatory molecules triggers conformational changes that affect the active sites.

Display cooperativity: small [S] changes - major activity changes.

Information transducers: signal changes activity or information shared by sites 2. Multiple forms of enzymes (isozymes).

times.

regulatory properties Used at distinct locations or Differ slightly in structure, in K m & V max values, and in 3. Reversible covalent modification. Activities altered by covalent attachment of modifying group, mostly a phosphoryl group 4. Protleolytic activation.

Irreversible conversion of an inactive form (zymogen) to an active enzyme

Aspartate transcarbamoylase reaction Committed step in pyrimidine synthesis: inhibited by end product CTP

CTP inhibits ATCase

CTP binds to regulatory subunits CTP stabilizes the T state

R and T states in equilibrium

ATCase displays sigmoidal kinetics Substrate binding to one active site converts enzyme to R state increasing their activity: active sites show cooperativity

Basis of sigmoidal curve R & T states equivalent to 2 enzymes with different K m s Cooperativity

Effect of CTP on ATCase kinetics CTP stabilizes the T state, curve shifts to right

Effect of ATP on ATCase kinetics ATP, allosteric activator , stabilizes R state, curve shifts to left

Oxygen delivery by hemoglobin, cooperativity enhanced 98 - 32 = 66% 63 - 25 = 38% Cooperativity enhances delivery 1.7 fold Partial pressure of oxygen

Heme group structure 4 linked pyrrole rings form a tetrapyrrole ring with a central iron atom.

side chains attached

Position of iron in deoxyhemoglobin Iron slightly outside porphyrin plane His (imidazole ring) binds 5th coordination site 6th site for O 2 binding

O 2 binding, conformational change Iron moves into plane, his is pulled along

Quaternary structure of hemoglobin Pair of identical alpha-beta dimers

Transition from T-to-R state in hemoglobin Interface most affected As O 2 binds, top pair rotate 15 o with respect to bottom pair

Oxygen affinity of fetal

v

maternal red blood cells Fetal Hgl does not bind 2,3-BPG, higher O 2 affinity Fetal hemoglobin tetramer has 2 alpha & 2 gama chains, Gene duplication

Isozymes of lactate dehydrogenase: glucose metabolism Rat heart LDH isozyme profile changes with development H (heart) isozyme (chain)= square, M (muscle) isozyme = circle

Tissue content of LDH Functional LDH is tetrameric, with different combinations of subunits possible.

H 4 (heart) has higher affinity for substrates than does M different allosteric inhibition by pyruvate 4 isozyme, H 4 H 3 M H 2 M 2 HM 3 M 4 Some isozymes in blood indicative of tissue damage, used for clinical diagnosis Increase in serum levels of H 4 relative to H 3 M, indicative of myocardial infraction (heart attack)

Examples of covalent modification

Phosphorylation widely used for regulation Gamma phosphoryl group

Some known protein kinases

Protein phosphotases Reverse the effects of kinases, catalyze hydrolytic removal of phosphoryl groups attached to proteins

Activation by proteolytic cleavage

Secretion of zymogens by acinar cell of pancreas Pancreas, one of the most active organs in synthesizing & secreting proteins Acinar cell stimulated by hormonal signal or nerve impulse, granule content released into duct to duodenum

Proteolytic activation of chymotrypsinogen Active enzyme generated by cleavage of a single specific peptide bond 3 chains linked by 2 interchain disulfide bonds, (A-B & B-C)

Conformations of chymotrypsinogen & chymotrypsin Electrostatic interaction between Asp 194 carboxylate & Ile 16  -amino group possible only in chymotrypsin, essential for activity

Zymogen activation by proteolytic cleavage Zymogens orange, active enzymes yellow Secreted by cells that line duodenum Digestive proteins of duodenum

Interaction of trypsin with its inhibitor Lys 15 & Asp 189 form salt bridge inside the active site