The Catalytic Role of the Active Site Aspartic Acid in Serine Proteases

Abstract

The role of the aspartic acid residue in the serine protease catalytic triad Asp, His, and Ser has been tested by replacing Asp 102 of trypsin with Asn by site-directed mutagenesis. The naturally occurring and mutant enzymes were produced in a heterologous expression system, purified to homogeneity, and characterized. At neutral p H the mutant enzyme activity with an ester substrate and with the Ser 195 -specific reagent diisopropylfluorophosphate is approximately 10 4 times less than that of the unmodified enzyme. In contrast to the dramatic loss in reactivity of Ser 195 , the mutant trypsin reacts with the His 57 -specific reagent, tosyl-L-lysine chloromethylketone, only five times less efficiently than the unmodified enzyme. Thus, the ability of His 57 to react with this affinity label is not severely compromised. The catalytic activity of the mutant enzyme increases with increasing p H so that at p H 10.2 the k cat is 6 percent that of trypsin. Kinetic analysis of this novel activity suggests this is due in part to participation of either a titratable base or of hydroxide ion in the catalytic mechanism. By demonstrating the importance of the aspartate residue in catalysis, especially at physiological p H, these experiments provide a rationalization for the evolutionary conservation of the catalytic triad.