SUBTILISIN BPN - ACTIVITY ON AN IMMOBILIZED SUBSTRATE

An immobilized substrate for serine proteases has been prepared, allowing for the simultaneous measurement of enzyme surface activity and hydrolytic activity at the interface. Subtilisin BPN' and a single site-directed variant (Y217L, native tyrosine 217 changed to leucine) adsorb onto the peptide-coated surface, hydrolyze the peptide, and release it into solution. Adsorption isotherms were measured for BPN' on the immobilized-substrate surface before and after hydrolysis. This enzyme/immobilized-substrate system has been modeled via the Michaelis-Menten equation and thereby provides kinetic parameters for comparison to the enzyme kinetic values reported for the same substrate free in solution. The overall enzyme catalytic efficiency is decreased when the substrate is immobilized. Comparing native BPN' with Y217L, which is catalytically more efficient (1.7X) on a soluble substrate, shows that the variant maintains superior efficiency (1.3X) on the insoluble form of the substrate. Finally, by expressing the data in terms of molecules per area, we are able to show that the hydrolysis rate for the surface-bound substrate increases as the enzyme surface concentration increases.