Cryoenzymology of penicillopepsin; with an appendix: mechanism of action of aspartyl proteinases.

Intrinsic spectral and kinetic properties of penicillopepsin and its action on N-acetylalanylalanyllysyl-p-nitrophenylalanylalanylalanine amide have been investigated at subzero temperatures in aqueous methanol and dimethyl sulfoxide solutions in an attempt to find evidence for or against a covalent mechanism in the catalyzed hydrolysis of peptide bonds. The study of fluorescence and circular dichroism spectra as a function of solvent concentrations gave no evidence for any solvent-induced structural effects at temperatures below the thermal denaturation transition. The effect of temperature on the intrinsic fluorescence of penicillopepsin in either 60% (v/v) methanol or 50% (v/v) dimethyl sulfoxide did not indicate any temperature-induced structural changes. On the other hand, Arrhenius plots for the hydrolysis reaction over the range 0 to -50 degrees C showed downward curvature. A probable explanation for this phenomenon is that the reduction in flexibility of the enzyme due to thermal and viscosity factors leads to the stabilization of a nonproductive conformation. The pH optima of kcat/Km are shifted from 5.1 in aqueous solvents to 5.6 in 60% methanol and to 6.6 in 50% dimethyl sulfoxide. Aqueous methanol caused small decreases of Km and of Kcat; the decrease in the latter was greater than that brought about by the decrease in the water concentration. In aqueous dimethyl sulfoxide, there was no detectable change in kcat up to 15%, but Km increased by more than an order of magnitude. Above 15%, only kcat/Km could be measured. No evidence for the accumulation of either covalent amino or covalent acyl intermediates was obtained when penicillopepsin was incubated at -70 degrees C in 67% methanol with several substrates. Although negative, these experiments do not rule out conclusively the involvement of covalent intermediates in penicillopepsin-catalyzed reactions.