Peptide substrate specificity of the membrane-bound metalloprotease of Leishmania.
The promastigote surface protease (PSP) of Leishmania is a neutral membrane-bound zinc enzyme. The protease has no exopeptidase activity and does not cleave a large selection of substrates with chromogenic and fluorogenic leaving groups at the P1' site. The substrate specificity of the enzyme was studied by using natural and synthetic peptides of known amino acid sequence. The identification of 11 cleavage sites indicates that the enzyme preferentially cleaves peptides at the amino side when hydrophobic residues are in the P1' site and basic amino acid residues in the P2' and P3' sites. In addition, tyrosine residues are commonly found at the P1 site. Hydrolysis is not, however, restricted to these residues. These results have allowed the synthesis of a model peptide, H2N-L-I-A-Y-L-K-K-A-T-COOH, which is cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 1.8 X 10(6) M-1 s-1. Furthermore, a synthetic nonapeptide overlapping the last four amino acids of the prosequence and the first five residues of mature PSP was found to be cleaved by the protease at the expected site to release the mature enzyme. This result suggests a possible autocatalytic mechanism for the activation of the protease. Finally, the hydroxamate-derivatized dipeptide Cbz-Tyr-Leu-NHOH was shown to inhibit PSP competitively with a KI of 17 microM.
Bouvier
J
J
Institut de Biochimie, Université de Lausanne, Epalinges, Switzerland.
Schneider
P
P
Etges
R
R
Bordier
C
C
D000595:Amino Acid Sequence; D000818:Animals; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007891:Leishmania / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism
D008565:Membrane Proteins; D010446:Peptide Fragments; D010455:Peptides; D015800:Protozoan Proteins; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania; D015032:Zinc