Interaction of papain with derivatives of phenylalanylglycinal.

The intrinsic tryptophan fluorescence of active papain is greatly enhanced upon the binding of the inhibitor acetyl+ phenylalanylglycinal, and of related compounds in which the acetyl group is replaced by benzyloxycarbonyl, benzyloxycarbonylglycyl, and benzyloxycarbonylglycylglycyl. This fluorescence enhancement is largely abolished when the active site cysteine residue of papain is blocked by mercuric ion or by conversion to the mixed disulfide with /3-mercaptoethanol. Although the total intrinsic tryptophan fluorescence of papain is reduced only slightly upon treatment of the enzyme with 4 molar equivalents of N-bromosuccinimide, the fluorescence enhancement evident upon binding of the glycinal derivatives is markedly diminished. In the light of the work of others showing that tryptophan177 of papain makes the major contribution to its total intrinsic fluorescence, and that tryptophan-69 is preferentially oxidized by N-bromosuccinimide, the conclusion is drawn that the fluorescence enhancement upon binding of the aldehyde inhibitors is largely a consequence of their interaction with tryptophan-69. A comparison has been made of the inhibitory capacity of the four glycinal derivatives used in this study. Whereas the acetyl derivative gave data consistent with earlier reports showing tight binding to form a 1:l enzyme’inhibitor complex, the compounds having a benzyloxycarbonyl group exhibited multisite (2:l) interaction with positive cooperativity. The latter compounds are approximately twice as effective in inhibiting papain as is the acetyl derivative. In connection with these experiments, acetyl+phenylalanylglycyl-p-nitroanilide was used as a standard substrate for spectrophotometric determination of papain kinetics, and its use for this purpose is recommended. Stopped flow fluorescence studies on the rate of increase in the intrinsic fluorescence of papain upon binding of the four glycinal derivatives in all cases showed at least two distinct steps. With acetyl+phenylalanylglycinal, a very rapid fluorescence increase is followed by a slower first order process whose observed rate constant increases hyperbolically with increasing inhibitor concentration. A value of 10 s-’ was estimated for the rate constant of the first order step at pH 6.5 and 25”. Comparable data with the