Analysis of matrix metalloproteinase triple-helical peptidase activity with substrates incorporating fluorogenic L- or D-amino acids.
Lauer-Fields JL., Kele P., Sui G., Nagase H., Leblanc RM., Fields GB.
The consequences of improper regulation of collagen turnover include diseases such as tumor cell metastasis and arthritis. Several fluorogenic triple-helical peptide (fTHP) substrates have been constructed presently to examine collagenolytic behavior. These substrates incorporate L- or D-2-amino-3-(7-methoxy-4-coumaryl)propionic acid (Amp) or L- or D-2-amino-3-(6,7-dimethoxy-4-coumaryl)propionic acid (Adp) as the fluorophore and N-2,4-dinitrophenyl (Dnp) as the quencher. The desired sequences were C6-(Gly-Pro-Hyp)5-Gly-Pro-[Amp/Adp]-Gly-Pro-Gln-Gly approximately Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Val-Arg-(Gly-Pro-Hyp)5-NH2. All four fTHPs formed stable triple-helices. Matrix metalloproteinase-2 (MMP-2) rates of hydrolysis for all fTHPs were considerably more rapid than corresponding MMP-1 rates. Evaluation of individual kinetic parameters indicated that MMP-2 bound to the fTHPs more efficiently than MMP-1. Comparison to a triple-helical substrate incorporating the same sequence but with a different fluorophore [Lys((7-methoxycoumarin-4-yl)acetyl); Lys(Mca)] demonstrated that the shorter side chain of Amp or Adp was better tolerated by MMP-1 and MMP-2. Adp may well be the fluorophore of choice for fTHPs, as (a) fTHPs incorporating Adp were obtained in significantly higher yields than the Amp-containing fTHPs, (b) Adp has a larger Stokes shift than either Amp or Lys(Mca) and thus has less chance of self-quenching, (c) Adp has a relatively high quantum yield, (d) the Adp/Dnp pair is compatible with multiwell plate reader formats, and (e) MMPs better tolerate Adp than Lys(Mca).