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Initial velocities of enzyme-catalyzed reactions

(experimentally observed at low [E];

multiple turnover conditions) [steady-state]

As [S] increases, the rate of product formation (velocity) increases

initial velocities are

the dashed lines

substrate depletion when time

points are taken too late

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At higher [S],

saturation is seen

At low [S], V increaseslinearly with [S]

Km= [S] when

V = (Vmax)

Effect of varying [S] on initial velocity, where [S] >> [E]

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CHYMOTRYPSIN-Cleaves proteins C-terminal to Tyr and Phe

amino acids

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Chymotrypsin improved activity because distal interactions

stabilize the transition state. Specificityversus peptides

possessing noncognate P1 residues also improves when the

substrates are larger.

Chymotrypsin: distal binding interactions

increase steady-state rate

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Chymotrypsin: rate-limiting step in the mechanism

Burst proves early fast step, later slow step; postulate 2-step

mechanism as shown. p-Nitrophenol is an ester analog

of a true peptide substrate and gives readout signal

[S] > [E]. Bothfirst turnover and steady state observed

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Lineweaver-Burke

replot of Michaelis-Menten

equation

1/V = Km/Vmax[1/S] + 1/Vmax

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Multiple substrates still apply steady-state; complex mechanisms

2 substrates; 2 products;

There may (or may not)

be an obligate order

of binding

Ping-pong kinetics