Substrates with Charged P1 Residues Are Efficiently Hydrolyzed by Serine Carboxypeptidases When S3−P1 Interactions Are Facilitated

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Abstract
The high activity of carboxypeptidase S1 with substrates having basic P1 residues is predicted to depend on the size of residue 312 in combination with the presence of a counter-charge in an α-helix above the S1 binding pocket. This hypothesis is tested by the construction of 32 mutant forms of carboxypeptidase Y that combines a reduction in size of residue 312 and the introduction of either a basic or an acidic residue at either position 241 or position 245. Kinetic characterization using substrates with Leu, Arg, Lys, Glu, or Asp in P1 demonstrates that most of these enzymes exhibit drastically altered catalytic properties. One mutant enzyme, N241D + W312L, hydrolyzes FA-Arg-Ala-OH with a kcat/KM value of 13 000 min-1 mM-1 corresponding to a 930-fold increase relative to the wild-type enzyme. This increased activity is due to an increase in kcat and is independent of ionic strength. The pH profile of kcat/KM exhibits an optimum around pH 5.5 similar to that observed for CPD-S1. Another mutant enzyme, L245R + W312S, hydrolyzes FA-Glu-Ala-OH and FA-Asp-Ala-OH with kcat/KM values of 5100 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 KM is observed. The activity of L245R + W312S is also independent of ionic strength and displays a virtually unaltered dependence on pH. The P1 substrate preference of these two mutant enzymes for Arg versus Asp differs 2.5 × 106-fold. values of single and double mutants demonstrate that the effects of reducing the size 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 be accounted for by each individual single mutation.