Topic%2003%20_%20Kinetics%20_%20F10

Topic%2003%20_%20Kinetics%20_%20F10 - Why study enzyme...

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Unformatted text preview: Why study enzyme kinetics? Compare enzymes from different sources, learn about the evolution of an enzyme, engineer better enzymes, determine how inhibitors affect enzymes, design better drugs that interact with enzymes. S P Kinetics How fast? Energetics Which way? ∆ G = – RT ln K eq Favorable direction has a – ∆ G Enzyme Kinetics simple 1 substrate : 1 product Energy ∆ G P S TS ‡ ∆ E + enz Two ways to measure enzyme activity: 1. Disappearance of S • Usually less accurate: hard to detect a small change in [S] when [S] is very high 2. Appearance of P • Usually more accurate: starting with [P] = 0, so detecting a small change in [P] is less questionable Kinetic measurements: +dP/dt = -dS/dt Spectrophotometric assays: apply Beer’s Law measure: +dAbs/dt or -dAbs/dt calculate: +d[P]/dt or -d[S]/dt v o = initial velocity of the reaction at a given [S] V max = maximum initial velocity at that [E] T K m (Michaelis Constant) = [S] where v o = ½ V max v o V max = [S] K m + [S] E + S ES E + P k 1 k –1 k cat Michaelis-Menten Kinetic Model Michaelis-Menton Kinetics Vo = Vmax·[S] = kcat·[Et]·[S] [S] >> [Et], saturation Vo = Vmax ~ kcat·[Et] [S] is not >> [Et], non-saturation Vo ≠ Vmax Estimation: if the [P] is ≤ 5% of the [S] at t o then valid assays conditions exist, Vo α [Et] Km + [S] Km + [S] Abs time v o v v V o > V > V Measure initial rates, v o, for enzymes V o: Reproducible Dependent on [E]t- relative measure of [E]t V [E] t ● ● ● ● ● ● Assay is valid in this region because v o is proportional to [E] t At saturating [S]: [S] >> [E] t [E] t v o v o ≈ V max and v o ∝ [E] t At lower, nonsaturating [S]: [E] t v o v o = V max ∝ [E] t K m + [S] [S] v o will be proportional to [Et] when the product has not reached a substantial concentration; “rule of thumb”: when approximately 5% or less of the substrate has been consumed Defining Units for Vo dAbs/dt = (d[P]/dt)(ε·l)...
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This note was uploaded on 11/21/2010 for the course MCB 120L 69059 taught by Professor Fairclough during the Fall '10 term at UC Davis.

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Topic%2003%20_%20Kinetics%20_%20F10 - Why study enzyme...

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