Chapter 8 Enzymes Significance of enzyme study: 1. Normal enzyme function is required for life...

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 enzymeat the active site

Enzymes affect reaction rates, not equilibria

E + S ES EP E + P

Ground stateTransition state vs. reaction intermediate Activation energyRate-limiting stepC12H22O11 + 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

Dihydrofolate reductaseNADP+

tetrahydrofolate

“lock and key” model

In reality

stickase

Induced fit

Lock and key

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/molFormation of a single weak interaction ~4-30 kJ/molBetween 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 factorsContributing to G , the barrier to reaction

Binding energy is used to overcome these barriers

1. The change in enthropy2. The solvation shell of H-bonded water3. The distortion of substrates4. The need for proper alignment of catalytic functional groups on the enzyme

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 + Pk1

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

Km = (k-1 + k2)/ k1 E + S ES E + Pk1

k-1

k2

if k2 << k-1 Km = k-1/ k1 = Kd Km relates to affinityif k2 >> k-1 Km = k2/ k1

if k2 ~ k-1

E + S ES E + Pk1

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

chymotrypsin

Irreversible inhibitorSuicide inactivatorMechanism-based inactivator

Enzyme activity is affected by pH

Enzyme-transition state complementarity

Transition-state analogsCatalytic antibodies

Reaction mechanisms illustrate principles

chymotrypsin

Amide nitrogens

AromaticSide chain

Steps in the hydrolytic cleavage of a peptide bound by chymotrypsin

Pre-steady state kineticevidence for an acyl-enzymeintermediate

Induced fit in hexokinase

The two-step reaction catalyzed byEnolase in glycolysis

P (orange)

O (blue)

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

+ Positive modulator- Negative modulator

S as a positive modulator

Vmax, Km

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

P-Ser14

GlucosePLP

Regulation of glycogen phosphorylase

Multiple phosphorylations allow exquisite regulatory control

OH

PO4

Proteinkinases

Proteinphosphatases

Multiple regulatory phosphorylations

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

-S-S-