Comparison of vertebrate collagenase and gelatinase using a new fluorogenic substrate peptide.
A fluorogenic substrate for vertebrate collagenase and gelatinase, Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2, was designed using structure-activity data obtained from studies with synthetic inhibitors and other peptide substrates of collagenase. Tryptophan fluorescence was efficiently quenched by the NH2-terminal dinitrophenyl group, presumably through resonance energy transfer. Increased fluorescence accompanied hydrolysis of the peptide by collagenase or gelatinase purified from culture medium of porcine synovial membranes or alkali-treated rabbit corneas. Amino acid analysis of the two product peptides showed that collagenase and gelatinase cleaved at the Gly-Leu bond. The peptide was an efficient substrate for both enzymes, with kcat/Km values of 5.4 microM-1 h-1 and 440 microM-1 h-1 (37 degrees C, pH 7.7) for collagenase and gelatinase, respectively. Under the same conditions, collagenase gave kcat/Km of about 46 microM-1 h-1 for type I collagen from calf skin. Since both enzymes exhibited similar Km values for the synthetic substrate (3 and 7 microM, respectively), the higher catalytic efficiency of gelatinase reflects predominantly an increase in kcat. Both enzymes were inhibited by HSCH2(R,S)CH[CH2CH(CH3)2]CO-L-Phe-L-Ala-NH2 in this assay (50% inhibition at 20 nM and less than 1 nM for collagenase and gelatinase, respectively). Soluble type I collagen was a competitive inhibitor of peptide hydrolysis by collagenase (KI = 0.8 microM) and exhibited mixed inhibition of gelatinase (KI = 0.3 microM).
Stack
M S
MS
Department of Biochemistry, University of Louisville School of Medicine, Kentucky 40292.
Gray
R D
RD
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000494:pharmacology; D003315:Cornea / Q000201:enzymology; D018093:Gelatinases; D006868:Hydrolysis; D007700:Kinetics; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D010434:Pepsin A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013552:Swine; D013583:Synovial Membrane / Q000201:enzymology
D000596:Amino Acids; D009842:Oligopeptides; D010446:Peptide Fragments; C058817:2,4-dinitrophenylprolyl-leucyl-glycyl-leucyl-tryptophyl-alanyl-argininamide; D003094:Collagen; D010447:Peptide Hydrolases; D010434:Pepsin A; D018093:Gelatinases; D003012:Microbial Collagenase