The substrate specificity of the collagenases from Clostridium histolyticum has been investigated by measuring the rates of hydrolysis of more than eighty peptides covering the P(,6) to P(,5)' binding sites of the enzymes. The choice of peptides was patterned after sequences found in the (alpha)1 and (alpha)2 chains of Type I collagen. To accomplish this, three continuously recording spectrophotometric assays have been developed. Each peptide contained either a 2-furanacryloyl or cinnamoyl group on position P(,2) or the 4-nitrophenylalanyl residue in position P(,1). Hydrolysis of the P(,1)-P(,1)' bond produces an absorbance change in thesse chromophores that has been used to quantitate the rate of hydrolysis of these peptides under first order conditions ({S}<<K(,m)) from which k(,cat)/K(,m) values have been calculated. The identity of the amino acid in all six positions (P(,3)-P(,3)') or elongation of the peptide to positions P(,6) or P(,5)' markedly influences the hydrolysis rates. This indicates that the enzymes have an extended active site. All six enzymes have a strict requirement for Gly in position P(,1)' and a preference for Gly in position P(,3). In general, class I enzymes (Bond and Van Wart, Biochemistry, 23, 3085, (1984)) prefer substrates with Pro or Ala in position P(,2)' and Hyp, Arg or Ala in position P(,3)'. Class II enzymes show a much broader specificity in positions P(,1)' through P(,3)' and are able to hydrolyze peptides containing all of the common triplets found in collagen. The complementary specificities between the classes of enzymes may explain the observed synergistic digestion of collagen by these enzymes. All six enzymes prefer hydrophobic residues in position P(,1) although the residues commonly found in collagen are hydrolyzed well. The enzymes show a preference for a correctly spaced Gly or Pro residue in the P(,3) to P(,1) / triplet and hydrolyze Gly-Pro, Gly-Ala or Ala-Pro sequences best. These studies indicate that the previously established rules for the specificity of these enzymes require modification. A new collagenase has been isolated (designated -collagenase) and purified to homogeneity. Its substrate specificity clearly indicates it is a class I enzyme. Finally, the mechanism of degradation of extended peptides has been investigated. / Source: Dissertation Abstracts International, Volume: 45-09, Section: B, page: 2907. / Thesis (Ph.D.)--The Florida State University, 1984.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_75430 |
| Contributors | STEINBRINK, DAVID RANDALL., Florida State University |
| Source Sets | Florida State University |
| Detected Language | English |
| Type | Text |
| Format | 228 p. |
| Rights | On campus use only. |
| Relation | Dissertation Abstracts International |
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