Cleavage specificity of human skin type IV collagenase (gelatinase). Identification of cleavage sites in type I gelatin, with confirmation using synthetic peptides.
Type IV collagenase (gelatinase) readily cleaves denatured collagen into very small peptides. Large cyanogen bromide fragments (25 kDa) of type I collagen are degraded at the same rate as the complete alpha-chain. A number of the gelatinolytic cleavage sites of alpha 1(I)CB7 and alpha 1(I)CB8, representing 50% of the collagen alpha-chain, were determined by sequence analysis of product peptides. In addition to the expected cleavage between glycine and hydrophobic residues, several other cleavage sites were identified. These sites were Gly-Glu, Gly-Asn, and Gly-Ser. Basic residues were found adjacent to the cleavage site in several cases. Hexapeptides containing these unexpected cleavage sites were synthesized, and Km and kcat values were determined. All but one of the Km values were in the submillimolar range, and turnover numbers for the peptides uncharged at the carboxyl terminus were on the order of 10,000/h. Of particular significance was the finding that hydroxyproline occurs 5 residues from the cleavage site in all carboxyl-terminal product peptides and also occurs 5 residues from the cleavage site in seven of nine amino-terminal product peptides. A requirement for hydroxyproline may be of importance in determining the specificity of this enzyme for denatured collagenous substrates.
Seltzer
J L
JL
Department of Dermatology, Washington University School of Medicine, St. Louis, Missouri 63110.
Akers
K T
KT
Weingarten
H
H
Grant
G A
GA
McCourt
D W
DW
Eisen
A Z
AZ
D000595:Amino Acid Sequence; D005780:Gelatin; D018093:Gelatinases; D006801:Humans; D007700:Kinetics; D003012:Microbial Collagenase / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis; D010434:Pepsin A / Q000378:metabolism*; D012867:Skin / Q000201:enzymology*; D013379:Substrate Specificity
D009842:Oligopeptides; D005780:Gelatin; D010434:Pepsin A; D018093:Gelatinases; D003012:Microbial Collagenase