The Leu-3 Residue of Serratia Marcescens Metalloprotease Inhibitor is Important in Inhibitory Activity and Binding with Serratia marcescens Metalloprotease

Abstract

Serratia marcescens metalloprotease inhibitor (SmaPI) is a proteinase inhibitor toward Serratia marcescens metalloprotease (SMP), In sequential deletion analysis of the N-terminal region of the SmaPI, SmaPIs starting at Ser-S and Leu-3 residues, respectively, had nearly a full inhibitory activity toward SMP. However, SmaPI starting at Ala-4 residue showed severely decreased inhibitory activity. Furthermore, kinetic analysis demonstrated that SmaPI starting at the Ala-4 residue had an inhibition constant for SMP approximately fourfold higher than that of wild-type SmaPI. The interactions of Leu-3 with SMP contribute 0.73 kcal mol(-1) to the overall stability of the SMP-SmaPI complex (8.44 kcal mol(-1)). To elucidate the detailed role of the Leu-3 residue in inhibitory activity of SmaPI, several site directed mutations were introduced. The inhibitory activities of Leu-3 mutants in which the Leu-3 has been converted to Ala, Asp, Gly, Ile, Lys, Phe, or Pro were correlated with the hydrophobicities of substituted amino acids. About 0.3 kcal mol(-1) is attributable to the side chain of the Leu-3 residue in the binding with SMP. From these results, it is suggested that (i) in contrast with the Erwinia chrysanthemi inhibitor, Gly-l and Ser-2 of SmaPI are not critical and (ii) the hydrophobicity of Leu-3 may be important in its inhibitory activity and binding with SMP. (C) 1998 Academic Press.