THE LEU-3 RESIDUE OF SERRATIA-MARCESCENS METALLOPROTEASE INHIBITOR ISIMPORTANT IN INHIBITORY ACTIVITY AND BINDING WITH SERRATIA-MARCESCENSMETALLOPROTEASE

Serratia marcescens metalloprotease inhibitor (SmaPI) is a proteinase inhibitor toward Serratia marcescens metalloprotease (SMP), In sequent ial deletion analysis of the N-terminal region of the SmaPI, SmaPIs st arting at Ser-S and Leu-3 residues, respectively, had nearly a full in hibitory activity toward SMP. However, SmaPI starting at Ala-4 residue showed severely decreased inhibitory activity. Furthermore, kinetic a nalysis demonstrated that SmaPI starting at the Ala-4 residue had an i nhibition constant for SMP approximately fourfold higher than that of wild-type SmaPI. The interactions of Leu-3 with SMP contribute 0.73 kc al mol(-1) to the overall stability of the SMP-SmaPI complex (8.44 kca l mol(-1)). To elucidate the detailed role of the Leu-3 residue in inh ibitory activity of SmaPI, several site directed mutations were introd uced. The inhibitory activities of Leu-3 mutants in which the Leu-3 ha s been converted to Ala, Asp, Gly, Ile, Lys, Phe, or Pro were correlat ed with the hydrophobicities of substituted amino acids. About 0.3 kca l mol(-1) is attributable to the side chain of the Leu-3 residue in th e binding with SMP. From these results, it is suggested that (i) in co ntrast with the Erwinia chrysanthemi inhibitor, Gly-l and Ser-2 of Sma PI are not critical and (ii) the hydrophobicity of Leu-3 may be import ant in its inhibitory activity and binding with SMP. (C) 1998 Academic Press.