Lecture Note 2

Lecture Note 2 - Experimental Determination of Vm and Km(or...

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1 Experimental Determination of V m and K m (or K m ’) Get activity from initial slope of rate vs. time curve. Have to have substrate IN EXCESS to get activity.
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3 Understanding K m K m is an intrinsic parameter. It is a constant derived from rate constants K m is, under true Michaelis-Menten conditions , an estimate of the dissociation constant of E from S, because at rapid equilibrium, Reversible reaction, dissociation constant is So small K m means tight substrate binding; high K m means weak substrate binding. K m equals to the substrate concentration [S] at which v =(1/2) v max 1 2 1 k k k K m + = ES S E + k 1 -1 [ ] 1 1 ] [ ] [ k k ES S E K d = = [ ] ] [ ] [ 1 1 ES k S E k = [ ] [] m K S S v v + = max
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4 Understanding v max •V max is asymptotically approached as substrate is increased, but never reached in reality. Turnover number , k cat (the catalytic center activity, indicating maximum reaction rate per active site per unit time), is defined as When the enzyme has only one active site , it describes number of substrates converted into products per enzyme, per unit time. ] [ 0 max E n v k cat = cat k E v k = = ] [ 0 max 2 n - # of active sites per enzyme molecule [ ] [] m K S S v v + = max
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6 Orders of the M-M Equation (2 nd , 1 st , 0 th orders) When substrate concentration is low, it is first order in [S], or Under the same low substrate concentration, [E 0 ] [E], so it is second order in [E] and [S] and k 2 / K m is called apparent second- order rate constant: When substrate concentration is high, the M-M equation is 0 th order in [S]: ] [ ] [ ] [ , ] [ , ] [ max max S K v K S S v v K K S so K S as m m m m m + = > + << max max ] [ ] [ ] [ v K S S v v K S as m m + = >> m m K E k K E k v ][S] [ ][S] [ 2 0 2
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7 Things to know Derive the Michaelis-Menten equation by Rapid Equilibrium Assumption and Quasi-Steady-State Assumption Define K m and V m Define K cat
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8 Plots of the MM Equation ( Be familiar with the plot ) Where K m corresponds to the horizontal axis when the vertical axis is half way of V max value. m K S S v v + = ] [ ] [ max
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9 How to calculate K M and V max from real data? Three kinds of plots : Lineweaver-Burk Plot Eadie-Hofstee Plot Hanes-Woolf plot
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10 Lineweaver-Burk Plot of MM Equation ( Be familiar with the plot ) ] [ 1 1 ] [ ] [ 1 ] [ ] [ max max max max S v K v S v K S v K S S v v m m m + = + = + = More conveniently, the MM equation can be plotted as a linear plot by rearranging the M-M expression below.
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11 β -Galactosidase β -Galactosidase
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12 Lineweaver-Burk Plot β -Galactosidase
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13 Eadie-Hofstee Plot of MM Equation ( Be familiar with the plot )
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Lecture Note 2 - Experimental Determination of Vm and Km(or...

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