COMPOUND ACTIVE SITE OF BACILLUS SUBTILIS NEUTRAL PROTEASE - SOME PROPERTIES OF 6 SUBSITES

To investigate the properties of each of the six subsites (S1-S3 and S1′-S3′) which constitute the compound active site of a neutral protease from Bacillus subtilis, a kinetic study was made using various synthetic peptides such as Z-A-(Gly)n-Leu-Ala and Z-Gly-Leu-(Gly)n-B(A or B = various d- and l-amino acid residues; n = 0, 1, and 2) as substrates. These peptides were all split at the peptide bond containing the amino group of l-leucine. Thus, in a kinetic study it was possible to examine the properties of each subsite when it was occupied by different synthetic peptides in which the kinds of A or B and the number n were varied in the peptide. The study indicated that these subsites all have both stereo- and sidechain-specificity, although the contribution of each subsite decreased as the distance from the catalytic site increased. The properties of these subsites differ qualitatively from each other: Subsite S1 is concerned only with binding, but subsite S1′ with both binding and catalysis, irrespective of the kind of amino acid residues. On the other hand, the other four subsites are concerned either with catalysis only or with both catalysis and binding, depending upon the kind of residues adjacent to the respective subsites. It was found further that both subsites S1 and S2′ produced a cooperative effect on the hydrolysis of a peptide bond recognized by subsite S1′. Thus, a very selective substrate Z-Phe-Leu-Ala was discovered, with a proteolytic coefficient about 1000 times higher than that of Z-Gly-Leu-NH2, which had previously been thought of as a specific substrate for the enzyme. © 1969.