The Leu-3 residue of Serratia marcescens metalloprotease inhibitor is important in inhibitory activity and binding with Serratia marcescens metalloprotease.
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-2 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-1 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.
Bae
K H
KH
College of Natural Sciences, Korea Advanced Institute of Science and Technology (KAIST) and Research Center for New Bio-Materials in Agriculture, 373-1 Kusong-dong, Taejon, Yusong-gu, 305-701, Korea.
Kim
I C
IC
Kim
K S
KS
Shin
Y C
YC
Byun
S M
SM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D016974:Dickeya chrysanthemi / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012706:Serratia marcescens / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D001426:Bacterial Proteins; D017931:DNA Primers; D011480:Protease Inhibitors; C096454:SmaPI protein, Serratia marcescens; D008666:Metalloendopeptidases; C080204:S. marcescens minor metalloproteinase; D007930:Leucine