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Spécificité de la protéase neutre de Micrococcus caseolyticus

1971; Wiley; Volume: 19; Issue: 1 Linguagem: Inglês

10.1111/j.1432-1033.1971.tb01286.x

1432-1033

M. Desmazeaud, J Hermier,

Protein Hydrolysis and Bioactive Peptides

The specificity of an exocellular neutral protease produced by Micrococcus caseolyticus has been investigated with amides, dipeptides mono‐and di‐substituted dipeptides and glucagon. This protease does not exhibit exopeptidase activity: it splits neither amides : Leu(NH 2 ), Phe(NH 2 ), Ala(NH 2 ), Val(NH 2 ), Ileu(NH 2 ), nor Gly‐Leu, Gly‐Phe, Gly‐Ala, nor Z‐Gly‐Leu, Z‐Gly‐Phe, Z‐Gly‐Phe, Z‐Gly‐Ala. There is no free amino acid in glucagon digests. Therefore this protease has to be considered as an endopeptidase. Moreover it splits neither AcTyrOEt or BzTyrOEt (chymotrypsin substrates) nor TosArgOMe or BzArgOEt (trypsin substrates). The results obtained with disubstituted dipeptides Z‐Gly‐X(NH 2 ) indicate that the peptide bonds which are hydrolyzed by the neutral protease were those involving amino groups of hydrophobic amino acids (Phe, Leu, Ala, Val, Ileu but not Trp) or of tyrosine. The highest rate of hydrolysis was obtained with Z‐Gly‐Phe (NH 2 ); it was 1.48 micromoles per minute per mg of protease at 37°C. The second residue (X′) involved in the bond by the carboxyl group in the series Z‐X′‐Phe(NH 2 ) has also an important effect on enzymic activity. So the rates of hydrolysis (in micromoles per min per mg of protease, at 37°), were: 2.01 for Z‐Ala‐Phe(NH 2 ), 1.48 for Z‐Gly‐Phe(NH 2 ) 0.25 for Z‐Leu‐Phe(NH 2 ), 0.146 for Z‐Ser‐Phe(NH 2 ), 0.140 for Bz‐Gly‐Leu(NH 2 ) and 0.122 for Z‐Gly‐Val(NH 2 ). This specificity has been confirmed with glucagon. Peptide bonds in which phenylalanine, leucine or alanine are involved by their amino group are rapidly split. The M. caseolyticus neutral protease displays a substrate specificity which is similar to that found for Pseudomonas aeruginosa or Streptomyces griseus . Thus the hydrophobic nature of the specificity of the protease of M. caseolyticus is not as absolute as that for the megateriopeptidase of Bacillus megaterium .