Chapter 8 Enzymes Significance of enzyme study: 1. Normal enzyme function is required for life maintenance 2. Medical treatment and diagnostic 3. Drug development

Introduction to Enzymes 1897 Eduard Buchner --- yeast extracts can ferment sugar to alcohol Frederick W. Kuhne --- the name “enzyme” 1926 James Sumner --- crystallization of urease John Northrop & Moses Kunitz --- crystallization of pepsin and trypsin J.B.S. Haldane --- treatise for “Enzymes” (weak-bonding interactions) Most enzymes are proteins

cofactor coenzyme prosthetic group holoenzyme apoenzyme (apoprotein)

Enzymes are classified by the reactions they catalyze

How enzymes work Binding of a substrate to an enzyme at the active site

Enzymes affect reaction rates, not equilibria E + S ES EP E + P Ground state Transition state vs. reaction intermediate Activation energy Rate-limiting step C12H22O11 + 12 O2 12 CO2 + 11 H2O

Reaction rates vs. Equilibria K’eq = [P]/[S] G’o = -RT ln K’eq V = k[S] = k [S1][S2] k = (k T/h)e-G /RT

A few principles explain the catalytic power and specificity of enzymes Binding energy (GB)--- the energy derived from enzyme-substrate interaction 1. Much of the catalytic power of enzymes is ultimately derived from the free energy released in forming multiple weak bonds and interactions between an enzyme and its substrate. This binding energy contributes to specificity as well as catalysis. 2. Weak interactions are optimized in the reaction transition state; enzyme active sites are complementary not to the substrate per se, but to the transition state through which substrates pass as they are converted into products during the course of an enzymatic reaction.

Weak interactions between enzyme and substrate are optimized in the transition state “lock and key” model Dihydrofolate reductase NADP+ tetrahydrofolate

In reality stickase Lock and key Induced fit

Role of binding energy in catalysis V = k [S1][S2] k = (k T/h)e-G /RT V can be increased 10 fold when -G decreased by 5.7 kJ/mol Formation of a single weak interaction ~4-30 kJ/mol Between E and S, GB ~60-100 kJ/mol

Binding energy vs. catalysis and specificity Specificity --- the ability of enzymes to discriminate between a substrate and a competing molecule. High specificity --- functional groups in the active site of enzyme arranged optimally to form a variety of weak interactions with a given substrate in the transition state

Physical and thermodynamic factors Contributing to G , the barrier to reaction 1. The change in enthropy 2. The solvation shell of H-bonded water 3. The distortion of substrates 4. The need for proper alignment of catalytic functional groups on the enzyme Binding energy is used to overcome these barriers

Rate enhancement by entropy reduction

Specific catalytic groups contribute to catalysis General acid-base catalysis

Amino acids in general acid-base catalysis 102 to 105 order of rate enhancement

Covalent catalysis A B A + B A B + X: A X + B A + X: + B H2O H2O Metal ion catalysis ionic interaction oxidation-reduction reactions

Enzyme kinetics as an approach to understanding mechanism Enzyme kinetics --- determination of the rate of the reaction and how it changes in response to changes in experimental parameters Fig. 8-11. Effect of substrate Concentration on the initial velocity of an enzyme-catalyzed reaction V0 (initial velocity) when [S]>>[E], t is short Vmax (maximum velocity) when [S] 

The relationship between substrate concentration and reaction rate can be expressed quantitatively E + S ES E + P k1 k-1 k2 V0 = k2[ES] Rate of ES formation = k1([Et]-[ES])[S] Rate of ES breakdown = k-1[ES] + k2[ES] Steady state assumption k1([Et]-[ES])[S] = k-1[ES] + k2[ES] k1[Et][S] - k1[ES][S] = (k-1 + k2)[ES] k1[Et][S] = (k1[S] + k-1 + k2)[ES] [ES] = k1[Et][S] / (k1[S] + k-1 + k2) divided by k1 [ES] = [Et][S] / {[S] + (k-1 + k2)/ k1} (k-1 + k2)/ k1 = is defined as Michaelis constant, Km [ES] = [Et][S] / ([S] + Km) V0 = k2[ES] = k2[Et][S] / ([S] + Km) Vmax = k2[Et] V0 = Vmax [S] / ([S] + Km) Michaelis-Menten equation

V0 = Vmax [S] / ([S] + Km) Michaelis-Menten equation When [S] = Km V0 = ½ Vmax

V0 = Vmax [S] / ([S] + Km) 1/V0 = Km /Vmax [S] + 1 /Vmax the double-reciprocal plot

Kinetic parameters are used to compare enzyme activities E + S ES E + P k1 k-1 k2 Km = (k-1 + k2)/ k1 if k2 << k-1 Km = k-1/ k1 = Kd Km relates to affinity if k2 >> k-1 Km = k2/ k1 if k2 ~ k-1

E + S ES E + P k1 k-1 k2 Vmax = k2[Et] kcat, the rate limiting of any enzyme-catalyzed reaction at saturation kcat = Vmax / [Et] (turnover number)

V0 = Vmax [S] / ([S] + Km) M-M equation kcat = Vmax / [Et] (turnover number) V0 = kcat [Et] [S] / ([S] + Km) when [S] << Km ([S] is usually low in cells) V0 = kcat [Et] [S] / Km ( kcat / Km , specific constant) kcat / Km has a upper limit (E and S diffuse together in aqueous solution) ~108 to 109 M-1S-1 catalytic perfection

Enzyme are subjected to inhibition Reversible vs. irreversible inhibition 1/V0 = Km /Vmax [S] + 1 /Vmax (the double-reciprocal plot) -1/Km

1/V0 = Km /Vmax [S] + 1 /Vmax 1/Vmax

1/V0 = Km /Vmax [S] + 1 /Vmax

Irreversible inhibition is an important tool in enzyme research and pharmacology Irreversible inhibitor Suicide inactivator Mechanism-based inactivator chymotrypsin

Enzyme activity is affected by pH

Enzyme-transition state complementarity Transition-state analogs Catalytic antibodies

Reaction mechanisms illustrate principles chymotrypsin

Amide nitrogens Aromatic Side chain

Steps in the hydrolytic cleavage of a peptide bound by chymotrypsin

Pre-steady state kinetic evidence for an acyl-enzyme intermediate

Induced fit in hexokinase

P (orange) O (blue) The two-step reaction catalyzed by Enolase in glycolysis

Regulatory enzymes Allosteric enzymes vs. allorsteric modulators Allosteric enzymes undergo conformational changes in response to modulator binding

Two views of the regulatory enzyme aspartate transcarbamoylase (12 subunits)

The regulatory step in many pathways is catalyzed by an allosteric enzyme Feedback inhibition

The kinetic properties of allosteric enzymes diverge from Michaelis-Menten behavior Vmax, Km S as a positive modulator + Positive modulator - Negative modulator

Some regulatory enzymes undergo reversible covalent modification

Phosphoryl groups affect the structure and catalytic activity of proteins Glycogen phosphorylase (Glucose)n + Pi (glucose)n-1 + glucose 1-phosphate

AMP PLP Glucose P-Ser14 Regulation of glycogen phosphorylase

Multiple phosphorylations allow exquisite regulatory control OH Protein phosphatases Protein kinases PO4

Multiple regulatory phosphorylations

Some types of regulation require proteolytic cleavage of an enzyme precursor --- zymogen -S-S-