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-