title: Analysis of matrix metalloproteinase triple-helical peptidase activity with substrates incorporating fluorogenic L- or D-amino acids.
abstract: 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).
authors.affiliation: Department of Chemistry and Biochemistry, Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431-0991, USA.
authors.forename: Janelle L
authors.initials: JL
authors.lastname: Lauer-Fields
authors.forename: Péter
authors.initials: P
authors.lastname: Kele
authors.forename: Guodong
authors.initials: G
authors.lastname: Sui
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nagase
authors.forename: Roger M
authors.initials: RM
authors.lastname: Leblanc
authors.forename: Gregg B
authors.initials: GB
authors.lastname: Fields
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007700:Kinetics; D020782:Matrix Metalloproteinases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D005456:Fluorescent Dyes; D020782:Matrix Metalloproteinases