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Evidence for a rate-limiting conformation change in the catalytic steps of the ficin and papain-catalysed hydrolyses of benzyloxycarbonyl-L-lysine p-nitrophenyl ester.

1 
The time-courses of ficin and papain-catalysed hydrolyses of carbobenzoxy-l-lysine p-nitro-phenyl ester (Z-Lys-ONp) have been followed employing enzyme and substrate concentrations ranging from situations where enzyme concentration is much greater than that of substrate ([E0] ≫ [S0]) to those where the concentration of Z-Lys-ONp greatly exceeds that of enzyme ([S0] ≫ [E0]). 
 
2 
When [S0] > [E0] a burst of p-nitrophenol release occurs within the first 3 ms of reaction. The amplitude of the burst is directly proportional to [E0] but independent of [S0]. These results are consistent with a simple three-step mechanism involving an acyl-enzyme intermediate. 
 
4 
When [E0] > [S0] with both enzymes, under conditions where the results with [S0] > [E0] indicate that the burst should be complete within 3 ms, an extra change in absorbance is observed with a half-life of about 20 ms, i.e. a first-order rate constant near 35 s−1. This rate constant is very close in value to the molecular activity of both enzymes. In this respect the reactions do not conform to the simple three-step mechanism. 
 
4 
The time-course of proton release during the reaction with [E0] ∼ [S0] closely parallels the time-course of p-nitrophenol release. The extent of the observed proton release is also close to that expected from theory. This result in conjunction with the foregoing is also inconsistent with a simple three-step mechanism. 
 
5 
The incorporation of 0.22 M hydroxylamine into reaction mixtures did not affect the rate of the enzyme-catalysed reaction under V conditions. This result is inconsistent with a rate-limiting step in catalysis involving the breakdown of a thioacyl-enzyme intermediate. 
 
6 
The foregoing observations lead to the conclusion that there must be an “extra” step in the hydrolysis of Z-Lys-ONp catalysed by these thiol proteases. Evidence is presented that this “extra”, rate-limiting catalytic step is a conformation change in the enzyme · Z-Lys-ONp complex, prior to the release of p-nitrophenol.