Identification of the rate-limiting step in serine proteinases from the effect of temperature on steady-state kinetics.

The effect of temperature on the steady-state kinetics of porcine pancreatic elastase can be used to determine whether acylation or deacylation is the rate-limiting step in catalysis. If acylation is rate-limiting, kcat and kcat/Km will show the same temperature dependence. If deacylation is rate-limiting, kcat will show a greater temperature dependence than kcat/Km. The temperature dependence of the steady-state kinetic parameters of t-Boc-Ala-Ala-Pro-Ala p-nitroanilide and N-acetyl-Ala-Ala-alpha-Aza-Ala p-nitrophenyl ester have been determined and are consistent with this prediction.