Specificity of megateriopeptidase: an amino-endopeptidase with hydrophobic characteristics

Abstract

An exocellular proteolytic enzyme produced by Bacillus megaterium and called megateriopeptidase has been isolated and its specificity has been investigated. Megateriopeptidase has no exopeptidase activity: it splits neither the usual substrates of carboxypeptidase A (Bz‐Gly‐Phe and Bz‐Gly‐Leu), nor those of carboxypeptidase B (Bz‐Gly‐Arg and Bz‐Gly‐Lys), nor the amides Gly(NH2), Ser(NH2), His(NH2), Arg(NH2), Met(NH2), Leu(NH2), Phe(NH2), Tyr(NH2), and Trp(NH2). This enzyme is strictly an endopeptidase because on the one hand there is no free amino acid in protein digests, on the other hand no cleavage of peptidic bonds adjacent to a free carboxyl or a free amino group is observed when megateriopeptidase is allowed to act on synthetic peptides. Megateriopeptidase splits neither trypsin substrates (BzArgOEt, TosArgOMe and BzArgNHN) nor chymotrypsin substrates (AcTyrOEt and BzTyrOEt).The specificity has first been investigated with natural polypeptides and proteins of known structure, by isolating peptide fragments with the fingerprinting technique. A tetrapeptide, an endecapeptide, insulin carboxymethylated B chain, glucagon and cytochrome c were used. Out of 24 cleaved bonds, 23 give peptides with a N‐terminal hydrophobic residue i. e. Phe, Leu, Ile, Val or Ala. The rate of hydrolysis of the various bonds as a function of time (from 1 min to 5 h 30 and of enzyme concentration (enzyme/substrate weight ratio 1/50 or 1/500) was roughly estimated in the case of glucagon and insulin B chain. Peptide bonds in which leucine is involved by its amino group are rapidly split, then those of phenylalanine and lastly those of other hydrophobic residues.The specificity has been confirmed with disubstituted dipeptides in which the amino and carboxyl groups are blocked. Eight derivatives of the series Z‐Gly‐X(NH2) in which X(NH2) is an amide residue were subjected to megateriopeptidase. Under the conditions used, Z‐Gly‐Leu(NH2) and Z‐Gly‐Phe(NH2) have been split but Z‐Gly‐Ala(NH2) has been weakly attacked and Z‐Gly‐Val(NH2) and Z‐Gly‐Ile(NH2) not at all as well as Z‐Gly‐Tyr‐(NH2), Z‐Gly‐Trp‐(NH2) and Z‐Gly‐Met(NH2). It seems therefore that the second residue involved in the bond by the carboxyl group may have a stimulating effect. Actually the rates of hydrolysis, given in μmoles/min per mg of enzyme acting at 37°, were 0.4 for Z‐Gly‐Phe(NH2), 1.2 for Z‐Ala‐Phe(NH2), 1.9 for Z‐Lys‐Phe(NH2), 2.6 for Z‐Ser‐Phe(NH2) and 3.6 for Z‐His‐Phe(NH2). The polar residues Ser and His appear to increase particularly the enzyme activity and the active centre could have a hydrophilic site spatially a neighbour of the hydrophobic main site.The general name of amino‐endopeptidases is suggested for enzymes splitting peptide bonds at the amino group of specific residues, in contrast to carboxy‐endopeptidases such as trypsin or chymotrypsin which split at the carboxyl group of specific residues. This type of enzyme has been found by other workers in several bacilli (B. subtilis, B. thermoproteolyticus) and presumably exists in invertebrates (Tenebrio molitor) and vertebrates (Crotalus atrox).