Enzyme Kinetics

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Enzyme Kinetics

• Chapter 6

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Kinetics

• Study of rxn rates, changes with experimental
  conds
• Simplest rxn S ?? P
• Rate measd by V velocity (M/sec)
• Depends on k, S

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Michaelis-Menten Kinetics

• Genl 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

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• Assume Vo condition -- S gtgtgt E
• Since S used up during rxn, cant 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 formn ES rate breakdown ES

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Experl Findings

• As incr S, V incrs linearly up to some max V
• At max V, little V incr regardless of S added

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M-M Relates E, S, P ? Experly Provable
Variables

• New constant
• KM (k2 k-1) / k1
• M-M eqn
• Vo (Vmax S) / (KM S)

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• Quantitative relationship between
• Initial velocity
• Max rate of rxn
• Initial S

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Experl 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

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Difficult to Determine Variables from M-M Plot

• Hard to measure small changes in V
• Use double reciprocal plot ? straight line
• Lineweaver-Burk (Box 6-1)

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KM

• 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

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• May describe affinity of E for S ONLY if k-1 gtgtgt
  k2
• Right half of rxn equation negligible
• KM k-1 / k1
• Describes rate formn, breakdown of ES
• Considered dissociation constant of ES complex
• Here, KM value indicates strength of binding E-S
• In real life, system more complex

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Other Kinetics Variables

• Turnover
• kcat
• S molecules converted ? P by 1 enz molecule per
  unit time
• Use when enz is fully satd w/ S

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Comparisons of Catalytic Abilities

• Optimum KM, kcat values for each E
• Use ratio to compare catalytic efficiencies
• Max efficiency at kcat / KM
  107 108 M-1 sec-1
• Velocity limited by E encounters w/ S
• Called Diffusion Controlled Limit

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Kinetics When gt1 Substrate

• Random order E can accept either S1 or S2 first
• Ordered mech 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

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Inhibition

• Cell controls catalysis in metabolic pathways
• Drugs, toxins alter catalysis by inhibn
• Used as tools to study mechs
• Irreversible
• Reversible
• Includes competitive, noncompetitive,
  uncompetitive

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Irreversible Inhibition

• Inhibitor binds tightly to enz
• Dissociates slowly or not at all
• Book example DIFP
• Includes suicide substrate inhibitors

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Reversible Inhibition

• Inhibitor may bind at active site or some distal
  site
• Binding reversible
• Temporarily inhibits E, S binding or proper rxn
• Can calculate KI

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• Competitive
• Appear as S
• Bind active site
• So compete w/ S for active site
• Overcome w/ incrd S
• Affects KM, not Vmax

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Reversible Inhibn (contd)

• Uncompetitive
• Binds only when S already bound (so ES complex)
• Does not bind active site
• ? Conforml change, E inactivated
• Not overcome w/ incrd S
• Affects both KM, Vmax
• Common when S1 S2

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Reversible Inhibn (contd)

• Noncompetitive (Mixed)
• S can be bound or not
• Does not bind active site
• Conforml change in E
• E inactd when I bound
• Decrd E avail for binding S, rxn catalysis
• Not overcome w/ incrd S
• Affects both KM, Vmax
• Common when S1 S2

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pH Affects Catalysis

• Optimum pH where max activity
• Aas impt to catalysis must maintain partic
  ionization
• Aas in other parts of enz impt to maintain
  folding, structure must also maintain partic
  ionization
• Can predict impt aas by activity changes at
  different pHs (use pKa info)

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