Abstract
The sequence specificity of human skin fibroblast collagenase has been investigated by measuring the rate of hydrolysis of 16 synthetic octapeptides covering the P4 through P4' subsites of the substrate. The choice of peptides was patterned after potential collagenase cleavage sites (those containing either the Gly-Leu-Ala or Gly-Ile-Ala sequences) found in types I, II, and III collagens. The initial rate of hydrolysis of the P1-P1' bond of each peptide has been measured by quantitating the concentration of amino groups produced upon cleavage after reaction with fluorescamine. The reactions have been carried out under first-order conditions ([S] much less than KM) and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P3 (Pro, Ala, Leu, or Asn), P2 (Gln, Leu, Hyp, Arg, Asp, or Val), P1' (Ile or Leu), and P4' (Gln, Thr, His, Ala, or Pro) all influence the hydrolysis rates. However, the differences in the relative rates observed for these octapeptides cannot in themselves explain why fibroblast collagenase hydrolyzes only the Gly-Leu and Gly-Ile bonds found at the cleavage site of native collagens. This supports the notion that the local structure of collagen is important in determining the location of the mammalian collagenase cleavage site.
Highlights
The sequence specificity ofhuman skin fibroblast bond at the cleavage sitemust lie in the influence of the collagenase has been investigated by measuring the surrounding residues in each a chain
The differencesin the relativerates observed for these octapeptides cannot in themselves explain why fibroblast collagenase hydrolyzes only the Gly-Leu and GlyIle bonds found at the cleavage siteof native collagens
It is of fundamental importance to determine whether it is the sequence specificity of tissue collagenasesor alocal conformational feature of the collagenase cleavage site that is responsible for this unique substrate specificity
Summary
EVIDENCE FOR THE ROLE OF COLLAGEN STRUCTUREINDETERMINING THE COLLAGENASE CLEAVAGE SITE*. These collagens are catabolized by the so-called tissue collagenases which have a characteristic and highly specific mode of action They cleave all three a chains of native types I, 11, and 111 collagens at a single locus by hydrolyzing the peptide bond followingthe Gly residue of the partial sequences Gly-[Ile or Leu]-[Ala or Leu] located approximately three-fourths from the NH2 terminus [3,4,5]. It non-cleavable, collagenase cleavage sites in native rat, calf, and chick type I and human typeI11 collagens.
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