Primary structure and inhibitory properties of a proteinase inhibitor produced by Streptomyces cacaoi

Abstract

Protein proteinase inhibitors showing sequence homology with Streptomyces subtilisin inhibitor (SSI) have been found to be distributed widely in Streptomyces species, and accordingly have been named SSI-like (SIL) proteins. SIL1 from S. cacaoi was the first of these proteins to be isolated and to be given a serial number. To study the structure-function relationship of SIL proteins, we determined the primary structure of SIL1 and measured its inhibitory activities. It was found to be composed of 110 amino acids and to exist in dimer form. The amino-acid sequence of SIL1 was unique among other characterized SIL proteins in having a one-residue deletion in two regions and a three-residue insertion in the flexible loop region. Sequence comparison indicated that SIL1 was distantly related to other members of the SSI family, and that amino-acid replacements had occurred not only on the surface of the SIL1 molecule but also in the β-sheet region. The reactive site of SIL1 was considered to be Arg 70-Glu 71 from sequence alignment with other SSI-family inhibitors. SIL1 inhibited subtilisin BPN′ strongly with an inhibitor constant ( K 1 of 2.8·10 −11 M, like other members of the SSI family possessing an Arg residue at the P1 site. In contrast, SIL1 exhibited weak inhibition toward trypsin with a K i value of 5.5 - 10 −8M, possibly as a consequence of insertion of the three residues in the flexible loop region near the reactive site. This contrast seems to be due to the difference in the subsite structure of the two proteinases.