HIGH-RESOLUTION CRYSTAL-STRUCTURE OF M-PROTEASE - PHYLOGENY AIDED ANALYSIS OF THE HIGH-ALKALINE ADAPTATION MECHANISM

M-protease is a subtilisin-family serine protease produced by an alkal iphilic Bacillus sp, strain, Optimal enzymatic activity of the protein occurs at pH 12.3. The crystal structure of M-protease (space group P 2(1)2(1)2(1), a = 62.3, b = 75.5, c = 47.2 Pi) has been refined to a c rystallographic a-factor of 17.2% at 1.5 Angstrom resolution, The alka line adaptation mechanism of the enzyme was analyzed, Molecular phylog eny construction was used to determine the amino acid substitutions th at occurred during the high-alkaline adaptation process, This analysis revealed a decrease in the number of negatively charged amino acids ( aspartic acid and glutamic acid) and lysine residues and an increase i n arginine and neutral hydrophilic amino acids (histidine, asparagine and glutamine) residues during the course of adaptation, These substit utions increased the isoelectric point of M-protease. Some of the acqu ired arginine residues form hydrogen bonds or ion pairs to combine bot h N- and C-terminal regions of M-protease, The substituted residues ar e localized to a hemisphere of the globular protein molecule where pos itional shifts of peptide segments, relative to those of the less alka liphilic subtilisin Carlsberg, are observed, The biased distribution a nd interactions caused by the substituted residues seem to be responsi ble for stabilization of the conformation in a high-alkaline condition .