10 - Enzyme Mechanisms.pdf

10 - Enzyme Mechanisms.pdf - Principles of Enzyme Catalysis...

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3/18/2017 1 1 Principles of Enzyme Catalysis Principles of Enzyme Catalysis I) Enzyme Kinetics II) Inhibition of Enzyme Activity III) Second-Order Reactions IV) Mechanisms of Enzyme Action V) Example: Serine Proteases VI) Enzyme Regulation 2 II) Inhibition of Enzyme Activity 3 1) Mix enzyme , substrate and inhibitor and follow the reaction 2) Determine V 0 at different substrate concentrations 3) Make plots of V 0 vs. [A] or 1/V 0 vs. 1/[A] 4) Repeat with different inhibitor concentration Inhibition type Rate equation Apparent K m Apparent V max None (α = 1, α’ = 1) 𝑉 0 = 𝑉 ?𝑎𝑥 [A] 𝐾 ? + [A] 𝐾 ? 𝑉 ?𝑎𝑥 Competetive ( ’ = 1) 𝑉 0 = 𝑉 ?𝑎𝑥 [A] 𝛼𝐾 ? + [A] 𝛼𝐾 ? 𝑉 ?𝑎𝑥 Uncompetetive ( = 1) 𝑉 0 = 𝑉 ?𝑎𝑥 [A] 𝐾 ? + 𝛼 [A] 𝑉 0 = 𝑉 ?𝑎𝑥 [A] 𝛼 𝐾 ? 𝛼 + [A] 𝐾 ? 𝛼 𝑉 ?𝑎𝑥 𝛼 𝑉 0 = 𝑉 𝑚𝑎𝑥 [𝐴] 1+ 𝐼 𝐾 𝐼 𝐾 𝑚 + 1+ 𝐼 𝐾 𝐼 [𝐴] or 𝑉 0 = 𝑉 𝑚𝑎𝑥 [𝐴] 𝛼𝐾 𝑚 +𝛼 [𝐴] 4
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3/18/2017 2 𝑉 0 = 𝑉 𝑚𝑎𝑥 [𝐴] 1+ 𝐼 𝐾 𝐼 𝐾 𝑚 + 1+ 𝐼 𝐾 𝐼 [𝐴] or 𝑉 0 = 𝑉 𝑚𝑎𝑥 [𝐴] 𝛼𝐾 𝑚 +𝛼 [𝐴] Inhibition type Rate equation Apparent K m Apparent V max Mixed Noncompetetive 𝑉 0 = 𝑉 ?𝑎𝑥 [A] 𝛼𝐾 ? + 𝛼 [A] 𝑉 0 = 𝑉 ?𝑎𝑥 [A] 𝛼 𝛼𝐾 ? 𝛼 + [A] 𝛼𝐾 ? 𝛼 𝑉 ?𝑎𝑥 𝛼 Pure Noncompetetive ( = ’) 𝑉 0 = 𝑉 ?𝑎𝑥 [A] 𝛼𝐾 ? + 𝛼[A] 𝑉 0 = 𝑉 ?𝑎𝑥 [A] 𝛼 𝐾 ? + [A] 𝐾 ? 𝑉 ?𝑎𝑥 𝛼 5 Inhibition Studies: Competetive ( α ’=1 ) 6 𝑉 0 = 𝑉 ?𝑎𝑥 [A] 𝛼𝐾 ? + [A] 𝑉 0 = 𝑉 ?𝑎𝑥 [?] 𝐾 ? + 𝛼′ [?] 𝑉 0 = 𝑉 ?𝑎𝑥 [?] 𝛼 𝐾 ? 𝛼 + [?] Inhibition Studies: Uncompetetive ( α =1) 7 𝑉 0 = 𝑉 ?𝑎𝑥 [?] 𝛼𝐾 ? + 𝛼′ [?] 𝑉 0 = 𝑉 ?𝑎𝑥 [?] 𝛼 𝛼𝐾 ? 𝛼 + [?] Inhibition Studies: Mixed Noncompetetive 8
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3/18/2017 3 𝑉 0 = 𝑉 ?𝑎𝑥 [?] 𝛼𝐾 ? + 𝛼[?] 𝑉 0 = 𝑉 ?𝑎𝑥 [?] 𝛼 𝐾 ? + [?] Inhibition Studies: Pure Noncompetetive ( α = α ) 9 Irreversible Inhibition Inhibitors that bind covalently with or destroy a functional group on an enzyme that is essential for the enzyme’s activity. Suicide Inactivator/Mechanism-Based Inactivator 10 III) Second-Order Reactions ? + ? ⇄ ? + ? Sequential/single-displacement reactions Both A and B are bound to the enzyme before the reaction can occur. “Ordered” or “random” Double-displacement (ping-pong) reactions Proceeds via formation of a covalently modified enzyme intermediate 11 Single-displacement/ sequential Second-Order Reactions 12
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