Characterization of the peptide substrate specificities of interstitial collagenase and 92-kDa gelatinase. Implications for substrate optimization.
The peptide substrate specificities of two matrix metalloproteinases (MMPs), interstitial collagenase (MMP-1), and 92-kDa gelatinase (MMP-9), have been examined. Starting with the parent substrate, Dnp-Pro-Leu-Gly approximately Leu-Trp-Ala-D-Arg-NH2, four separate substrate mixtures were synthesized at subsites P2(Leu) through P2'(Trp). These mixtures contained either naturally occurring L-amino acids, D-amino acids, or either of two distinct sets of miscellaneous amino acids. Combined, these mixtures gave 88 unique substitutions at each position and, over the four subsites, represented 352 potential substrates. Optimal substrates were identified using a combined high performance liquid chromatography/mass spectrometry analysis as previously reported. The results gave an extended profile of the substrate specificities for both MMP-1 and MMP-9 at subsites P2(Leu) through P2'(Trp). Using the data obtained from the mapping, a new peptide substrate, Dnp-Pro-Cha-Abu approximately Smc-His-Ala-D-Arg-NH2 (where Dnp is 2,4-dinitrophenyl, Cha is cyclohexylalanine, Abu is alpha-aminobutyric acid, and Smc is S-methylcysteine) was designed and characterized. This peptide showed a 36-fold improvement in turnover (kcat/Km) versus the parent substrate by interstitial collagenase. In addition, some collagenase subsite specificities described here were found to be different from those previously reported. Experimental data show that the observed selectivity is dependent on the original peptide template employed, which has broader implications for substrate specificity studies.
McGeehan
G M
GM
Glaxo Inc. Research Institute, Research Triangle Park, North Carolina 27709.
Bickett
D M
DM
Green
M
M
Kassel
D
D
Wiseman
J S
JS
Berman
J
J
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D017364:Collagenases / Q000378:metabolism*; D005109:Extracellular Matrix / Q000201:enzymology; D005347:Fibroblasts / Q000201:enzymology; D018093:Gelatinases / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D017364:Collagenases; D018093:Gelatinases