Primary structure of Paim I, an .alpha.-amylase inhibitor from Streptomyces corchorushii, as determined by the combination of Edman degradation and fast atom bombardment mass spectrometry

Abstract

Paim I, a protein alpha-amylase inhibitor, inhibits animal alpha-amylases from pig, dog, cow, horse, etc. but has no activity against human salivary and pancreatic amylases. The primary structure of Paim I has been determined by Edman degradation and fast atom bombardment mass spectrometry (FABMS). This protein is a single-chain polypeptide of 73 amino acid residues with a calculated molecular weight from the sequence data of 7415.3 (monoisotopic molecular weight) and 7420.2 (average molecular weight). The sequencing strategy chosen for Paim I consists of four steps. First, the accurate molecular weights of the intact and tetra-S-carboxymethylated Paim I are determined by fast atom bombardment mass spectrometry. Second, the primary fragments generated by Staphylococcus aureus V8 protease are isolated by reversed-phase high-performance liquid chromatography. The molecular weights of these subpeptides are determined by FABMS. The peptides that must be sequenced are selected by the molecular weights of these subpeptides and the tetra-S-carboxymethylated Paim I. Third, these subpeptides and the whole protein are sequenced by automated Edman degradation. Finally, the primary structure of tetra-S-carboxymethylated Paim I is confirmed by the combination of tryptic, chymotryptic, and S. aureus V8 protease digestion and FABMS. The sequence of Paim I is compared with those of Haim II, Hoe-467A, Z-2685, and AI-3688 because they have different alpha-amylase inhibition spectra against mammalian alpha-amylases but belong to a family of related proteins.

Carbohydrate and Protein-based Inhibitors of Porcine Pancreatic α-Amylase: Structure Analysis and Comparison of Their Binding Characteristics

“…This might also be the reason why Streptomyces has ''invented'' a second, proteinaceous class of a-amylase inhibitors such as e.g. Tendamistat (Aschauer et al, 1983), Paim (Hirayama et al, 1987), Haim (Murao et al, 1980), Z-2685 (Oeding et al, 1979), AI-409 (Katsuyama et al, 1992), and T-76 (Sumitani et al, 1993). These interact with roughly the same residues in the active site region as the oligosaccharide derived inhibitors, but make use of additional binding regions further away from the active site.…”

Tendamistat (12–26) fragment

“…Since the Argl9 residue is in a special region of the polypeptidic chain, flanked by two aromatic residues and located in a well-conserved j-turn segment of the chain [24], we checked the possibility of modifying the stability of the structure by making mutations in that region. Trp was changed to Ser, a non-aromatic residue with a high turn probability in any position within the turn [36], with the idea in mind of removing the aromatic side chain, but maintaining the turn tendency of the region.…”

“…2). Third, the sequence at the turn is very well conserved in microbial a-amylase inhibitors [24] and when modified, the biological function is lost [25]; this may be a consequence of a loss of structure in that region of the chain. In this paper, the conformational properties in aqueous solution of three peptides corresponding to the 12 -26- …”

A determination of the positions of disulphide bonds in Paim I, α-amylase inhibitor fromStreptomyces corchorushii, using fast atom bombardment mass spectrometry

“…5). 9 In addition to that, the location of the disulphide bridges and amino acid sequence of active site Trp( 15)-Arg-Tyr(l7) were proved to be identical in all inhibitors. Therefore, the three-dimensional structures of these inhibitors have been considered to be However, the activity of Paim I is somewhat different from that of other inhibitors.…”