Fluorescent oligopeptide substrates for kinetic characterization of the specificity of Astacus protease.
The design of fluorescent N-dansylated oligopeptides based on the tubulin cleavage pattern by Astacus protease yields substrates that are turned over up to 10(5) times faster than those presently available. On the basis of this study, an optimal substrate for Astacus protease contains seven or more amino acids and minimally requires at least five amino acids. Direct examination of the formation and breakdown of the ES complex shows its formation occurs within milliseconds at 25 degrees C. The best heptapeptide substrate, Dns-Pro-Lys-Arg-Ala-Pro-Trp-Val, is cleaved only between the Arg-Ala (P1-P1') bond with kinetic parameters kcat = 380 s-1 and Km = 3.7 x 10(-4) M. The presence of Lys or Arg in the P1 and P2 positions yields high-turnover substrates. In the P3 position, the enzyme prefers Pro greater than Val greater than Leu greater than Ala greater than Gly, following the same order of preference seen in the tubulin cleavage pattern. Substitution of Leu for Ala in P1' and of Ser for Pro in P2' decreases activity by 10(5)- and 10(2)-fold, respectively. In position P3', substitution of Trp for Leu leaves the activity unaltered. However, introduction of the Trp fluorophore greatly enhances the sensitivity of the assay due to a 10-fold increase in indole fluorescence for cleavage of any peptide bond between the tryptophan and the dansyl group. Such an energy-transfer-based assay should have widespread use for detection of neutral proteases.(ABSTRACT TRUNCATED AT 250 WORDS)
Stöcker
W
W
Department of Physiology, University of Heidelberg, FRG.
Ng
M
M
Auld
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DS
D000595:Amino Acid Sequence; D000818:Animals; D003400:Astacoidea / Q000201:enzymology*; D001665:Binding Sites; D019485:Bone Morphogenetic Proteins; D003619:Dansyl Compounds / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D004735:Energy Transfer; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D011506:Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry; D014404:Tubulin / Q000737:chemistry; D015032:Zinc / Q000378:metabolism
D019485:Bone Morphogenetic Proteins; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D011506:Proteins; D014404:Tubulin; D014364:Tryptophan; D010450:Endopeptidases; D008666:Metalloendopeptidases; D015032:Zinc