Cleavage specificity of type IV collagenase (gelatinase) from human skin. Use of synthetic peptides as model substrates.
Type IV collagenase (gelatinase) has a marked substrate specificity for denatured collagen (gelatin). Cleavage site specificity of type IV collagenase from human skin was determined using small collagenous peptides with varied sequences around Gly-Leu or Gly-Ile. Type IV collagenase showed essentially the same order of preference for the peptide substrates as did interstitial collagenase. Both required a peptide with a minimum of six amino acid residues to demonstrate significant gelatinolytic activity and were able to cleave uncharged molecules more rapidly than charged molecules. the repeating Gly-X-Y-Gly sequence of collagen is not an absolute requirement for either enzyme since both digested AcPro-Leu-Gly-Ile-Leu-Ala-Ala-OC2H5 at 70% of the rate of the best substrate peptide, AcPro-Leu-Gly-Leu-Leu-Gly-OC2H5. Km and kcat (Vmax) values were determined for several of the peptides and for the native substrate. Turnover numbers with type IV collagenase were similar to those with interstitial collagenase (Weingarten, H., Martin, R., and Feder, J. (1985) Biochemistry 24, 6730-6734). However, the Km for all peptides investigated was approximately 10-fold lower for type IV collagenase than for interstitial collagenase. Because type IV collagenase does not cleave helical interstitial collagens, the data support the conclusion that secondary structure determines whether the peptide bond can be hydrolyzed at any potential cleavage site.
Seltzer
J L
JL
Department of Dermatology, Washington University School of Medicine, St. Louis, Missouri 63110.
Weingarten
H
H
Akers
K T
KT
Eschbach
M L
ML
Grant
G A
GA
Eisen
A Z
AZ
D000595:Amino Acid Sequence; D003488:Cyanogen Bromide; D018093:Gelatinases; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010434:Pepsin A / Q000302:isolation & purification / Q000378:metabolism*; D012867:Skin / Q000201:enzymology*; D013379:Substrate Specificity
D009842:Oligopeptides; D010434:Pepsin A; D018093:Gelatinases; D003488:Cyanogen Bromide