On the Reactive Site of Streptomyces Subtilisin Inhibitor

Abstract

The reactive of Streptomyces subtilisin inhibitor (SSI) was investigated by chymotryptic cleavage of the reactive site peptide bond and its resynthesis by inhibitor-subtilisin complex formation. The results show that the reactive site peptide bond of the inhibitor is Met(73)-Val(74) and that subtilisin specifically cleaved the Leu(6)-Tyr(7) bond of SSI upon complex formation.

Enzymatic oxidation of the bifunctional wheat inhibitor of subtilisin and endogenous α‐amylase

“…On the basis of the aforesaid evidence, we may conclude that the reactive site of the bifunctional wheat inhibitor includes Met34~la 35. It is noteworthy that the methionine residue at the P1 position was detected in the reactive sites of a number of protease inhibitors of microbial serine proteinases [16][17][18]. As follows from the results obtained, the ability of methionine residues to be enzymatically oxidized under mild conditions is a possible way to regulate proteolytic activity.…”

Effect of Enzymic Hydrolysis of the Scissile Bond on the Secondary Structure of a Dimeric Globular Protein

“…These results are consistent with the fact that the nicked bond is in the loop of the second disulfide bond as shown in Figure 1. 13 An elution profile of the native SSI* on a Sephadex G-100 column corresponded to that of the SSI on the same column. Furthermore, the thermal denaturation of SSI* authentic sample was examined and the same temperature profile of CD spectra was obtained.…”

“…25 have shown the CD spectrum of the modified (nicked) soybean trypsin inhibitor (STI*) to be almost identical with that of the intact one (STI) and the stoichiometry of their reactions has been extensively studied. 26 • 27 The reactive bonds of SSI 13 and STI 26 where f and C 0 are the denatured fraction obtained from the denaturation profile ( Figure 6) and the total protein concentration, respectively. The enthalpy change (11H) for this denaturation is defined as follows,…”

“…11 • 12 SSI* is a reaction intermediate formed in the protein-protein interaction. Since the reactive site is located in a cyclic peptide bonds linked by a disulfide bond (Cys 71-Cys 101) 13 , the modification of SSI leaves the protein molecule in one unit as schematically shown in Figure 1 drawn based on the X-ray study. 3 Denaturation of SSI* has not been studied except for a preliminary fluorometric study on the environment of a Trp residue in the subunit.…”

Modification of API-2c and Its Reactive Site

“…6 ) Recently enzymatic modification of the reactive site of S-SI has been reported by Omichi et aZ. 14 ) They elucidated that the MetVal bond is the reactive site. Although API-2c resembles the other microbial inhibitors such as AP_I,15) S-SI and Plasminostreptin l6 ) in many features, including amino acid composition, molecular weight and selfassociation behavior, API -2c is characteristic in that it contains an isoleucine residue in the molecule.…”