Enzyme Kinetics Chapter 8. Kinetics Study of rxn rates, changes with changes in experimental...
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Transcript of Enzyme Kinetics Chapter 8. Kinetics Study of rxn rates, changes with changes in experimental...
Enzyme Kinetics
Chapter 8
Kinetics
• Study of rxn rates, changes with changes in experimental conditions
• Simplest rxn: S <==> P
– Rate meas’d by V = velocity (M/sec)
– Depends on k, [S]
Michaelis-Menten
• Gen’l theory rxn rate w/ enzymatic catalysis
• Add E, ES to rxn:
E + S <==> ES <==> E + P
• Assume little reverse rxn E + P ES
So E + S <==> ES E + P
• Assign rate constants k1, k-1, k2
• Assume Vo condition: [S] >>> [E]
– Since S used up during rxn, can’t be limiting
• Assume:
– All E goes to ES
• Assume fixed amt enzyme
– If all E ES, will see max rate of P formed
– At steady state, rate form’n ES = rate breakdown ES
Exper’l findings:
– As incr [S], V incr’s linearly up to some max V
– At max V, little V incr regardless of [S] added
Fig. 8-11
M-M relates [E], [S], [P] exper’ly provable variables• New constant: KM = (k2 + k-1) / k1
• M-M eq’n:
• Vo = (Vmax [S]) / (KM + [S])
• Quantitative relationship between
– Initial velocity
– Max rate of rxn
– Initial [S]
Exper’l definition of KM
• At ½ Vmax (substitute ½ Vmax for Vo)
• Divide by Vmax
• Solve for KM
• KM = [S]
• So when Vo = ½ Vmax , KM = [S]
Fig. 8-12
Difficult to determine variables from M-M plot
• Hard to measure small changes in V
• Use double reciprocal plot straight line
• Lineweaver-Burk (Box 8-1)
Box 8-1
KM (Table 8-6)
• [S] at which ½ enz active sites filled
• Related to rate constants
• In living cells, value close to [S] for that E
– Commonly enz active sites NOT saturated w/ S
• May describe affinity of E for S ONLY if k-1 >>> k2
– Right half of rxn equation negligible
– KM = k-1 / k1
– Describes rate form’n, breakdown of ES
– Here, KM value indicates strength of binding E-S
– In real life, system is more complex
Table 8-6
Other kinetics variables (Table 8-7)
• Turnover #
– # S molecules converted P by 1 enz molecule per unit time
– Use when enz is fully sat’d w/ S
– Equals k2
– Can calc from Vmax if know [ET]
Other kinetics variables (cont’d)
• kcat
– Max catalytic rate for E when S saturating
– Equivalent to k of rate limiting step
– For M-M ( E + S <==> ES <==> E + P ), kcat = k2
– Can be complex
– Book = turnover #
Table 8-7
Comparisons of catalytic abilities• Optimum KM, kcat values for each E
• Use ratio to compare catalytic efficiencies
• Max efficiency at kcat / KM = 108 – 109 M-1 sec-1
– Velocity limited by E encounters w/ S
– Called Diffusion Controlled Limit
Table 8-8
Kinetics when > 1 substrate
• Random order = E can accept either S1 or S2 first
• Ordered mechanism = E must accept S1 first, before S2 can bind
• Double displacement (or ping-pong) = S1 must bind and P1 must be released before S2 can bind and P2 is released
Fig. 8-13
Fig. 8-13 (cont’d)
Inhibition
• Used by cell to control catalysis in metabolic pathways
• Used to alter catalysis by drugs, toxins
• Used as tools to study mechanisms
• Irreversible
• Reversible
– Includes competitive, noncompetitive, uncompetitive
Irreversible inhibition
• Inhibitor binds tightly to enz
• Dissociates slowly or not at all
• Book example: DIFP
• Includes suicide substrate inhibitors
Fig. 8-16
Reversible inhibition• Inhibitor may bind at active site or some distal
site
• Binding is reversible
• Temporarily inhibits E, S binding or proper rxn
• Can calculate KI
• Competitive
– “Appear as S”
– Bind active site
• So compete w/ S for active site
– Overcome w/ incr’d [S]
– Affects KM, not Vmax
Fig. 8-15
Reversible inhibition (cont’d)
• Noncompetitive (Mixed)
– When S bound or not
– Bind at site away from active site
– Causes conform’l change in E
– E inactivated when I bound
– Decr’d E avail for binding S, rxn catalysis
– Not overcome w/ incr’d [S]
– Affects both KM, Vmax
– Common when S1 + S2
Fig. 8-15 (cont’d)
Reversible inhibition (cont’d)
• Uncompetitive
– Binds only when S already bound (so ES complex)
– Bind at site away from active site
– Causes conform’l change, E inactivated
– Not overcome w/ incr’d [S]
– Affects both KM, Vmax
– Common when S1 + S2
Fig. 8-15 (cont’d)
Effect of pH on catalysis
• Optimum pH where maximal activity
• Aa’s impt to catalysis must maintain particular ionization
• Aa’s in other parts of enz impt to maintain folding, structure must also maintain partic. ionization
• Can predict impt aa’s by activity changes at different pH’s (use pKa info)
Fig. 8-17