Kinetics of nitroanilide cleavage by astacin.
The investigation of the catalytic properties of astacin, a zinc-endopeptidase from the crayfish Astacus astacus L., has gained importance, because the enzyme represents a novel, structurally distinct family of metalloproteinases which also includes a human bone morphogenetic protein (BMP1). Astacin releases nitroaniline from succinyl-alanyl-alanyl-alanyl-4-nitroanilide (Suc-Ala-Ala-Ala-pNA), a substrate originally designed for pancreatic elastase. This activity was unexpected since only few metalloproteinases cleave small nitroanilide substrates, and, moreover, the primary specificity of astacin toward protein substrates is determined by short, uncharged amino-acid sidechains in the P'1-position, i.e. the new N-terminus after cleavage. The specificity constants, kcat/Km, for the release of nitroaniline from substrates of the general structure Suc-Alan-pNA (n = 2, 3, 5) and Alan-pNA (n = 1, 2, 3) increase with the number of alanine residues. The longest peptide, Suc-Ala(-)-Ala-Ala-Ala-Ala-pNA, is the only one out of eleven substrates used in this study, which is cleaved at two positions by astacin. The first cleavage yields Suc-Ala(-)-Ala and Ala-Ala-Ala-pNA. From the resulting C-terminal fragment, Ala-Ala-Ala-pNA, a second cut releases nitroaniline. The 1200-fold higher specificity constant observed for the first as compared to the second cleavage in Suc-Ala-Ala-Ala-Ala-Ala-pNA reflects the preference of astacin for true peptide bonds and also the importance of a minimum length of the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
Stöcker
W
W
Zoologisches Institut, Universität Heidelberg, Fachrichtung Physiologie.
Sauer
B
B
Zwilling
R
R
D000595:Amino Acid Sequence; D000813:Anilides / Q000378:metabolism*; D000818:Animals; D003400:Astacoidea; D002852:Chromatography, Ion Exchange; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008666:Metalloendopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009574:Nitro Compounds / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D013379:Substrate Specificity
D000813:Anilides; D007527:Isoenzymes; D009574:Nitro Compounds; D009842:Oligopeptides; D008666:Metalloendopeptidases; C071288:astacin