SUBSTRATES WITH CHARGED P-1 RESIDUES ARE EFFICIENTLY HYDROLYZED BY SERINE CARBOXYPEPTIDASES WHEN S-3-P-1 INTERACTIONS ARE FACILITATED

The high activity of carboxypeptidase S1 with substrates having basic P-1 residues is predicted to depend on the size of residue 312 in comb ination with the presence of a counter-charge in an a-helix above the S-1 binding pocket. This hypothesis is tested by the construction of 3 2 mutant forms of carboxypeptidase Y that combines a reduction in size of residue 312 and the introduction of either a basic or an acidic re sidue at either position 241 or position 245. Kinetic characterization using substrates with Leu, Arg, Lys, Glu, or Asp in P-1 demonstrates that most of these enzymes exhibit drastically altered catalytic prope rties. One mutant enzyme, N241D + W312L, hydrolyzes FA-Arg-Ala-OH with a k(cat)/K-M value of 13 000 min(-1) mM(-1) corresponding to a 930-fo ld increase relative to the wild-type enzyme. This increased activity is due to an increase in k(cat) and is independent of ionic strength. The pH profile of k(cat)/K-M exhibits an optimum around pH 5.5 similar to that observed for CPD-S1. Another mutant enzyme, L245R + W312S, hy drolyzes FA-Glu-Ala-OH and FA-Asp-Ala-OH with k(cat)/K-M values of 510 0 and 5300 min(-1) mM(-1), respectively, corresponding to 120 and 170- fold increases relative to wild-type values. With the latter substrate , a 280-fold reduction of K-M is observed. The activity of L245R + W31 2S is also independent of ionic strength and displays a virtually unal tered dependence on pH. The P-1 substrate preference of these two muta nt enzymes for Arg versus Asp differs 2.5 x 10(6)-fold. Delta Delta G( T)(double dagger). values of single and double mutants demonstrate tha t the effects of reducing the sire of Trp312 and introducing a charged residue at position 241 or 245 in some cases exceed 100% additivity. Thus, the double mutant enzyme gains more activation energy than can b e accounted for by each individual single mutation.