Kinetic Analysis of Matrix Metalloproteinase Activity Using Fluorogenic Triple-Helical Substrates

• Published in: Biochemistry (Easton) Vol. 40; no. 19; pp. 5795 - 5803
• Main Authors: Lauer-Fields, Janelle L, Broder, Todd, Sritharan, Thilaka, Chung, Linda, Nagase, Hideaki, Fields, Gregg B
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 15.05.2001
• Subjects: Amino Acid Sequence; Animals; Cells, Cultured; Collagen - metabolism; Collagenases - metabolism; Fluorescent Dyes - metabolism; Humans; Hydrolysis; Kinetics; Matrix Metalloproteinase 1 - metabolism; Matrix Metalloproteinase 13; Matrix Metalloproteinase 2 - metabolism; Matrix Metalloproteinase 3 - metabolism; Matrix Metalloproteinases - metabolism; Molecular Sequence Data; Peptide Fragments - chemical synthesis; Peptide Fragments - metabolism; Protein Structure, Secondary; Rabbits; Substrate Specificity; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0101190

Matrix metalloproteinase (MMP) family members are involved in the physiological remodeling of tissues and embryonic development as well as pathological destruction of extracellular matrix components. To study the mechanisms of MMP action on collagenous substrates, we have constructed homotrimeric, fluorogenic triple-helical peptide (THP) models of the MMP-1 cleavage site in type II collagen. The substrates were designed to incorporate the fluorophore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitrophenyl (Dnp) in the P5 and P5‘ positions, respectively. In addition, Arg was incorporated in the P2‘ and P8‘ positions to enhance enzyme activity and improve substrate solubility. The desired sequences were Gly-Pro-Lys(Mca)-Gly-Pro-Gln-Gly∼Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Ile/Val-Arg. Two fluorogenic substrates were prepared, one using a covalent branching protocol (fTHP-1) and one using a peptide self-assembly approach (fTHP-3). An analogous single-stranded substrate (fSSP-3) was also synthesized. Both THPs were hydrolyzed by MMP-1 at the Gly∼Leu bond, analogous to the bond cleaved in the native collagen. The individual kinetic parameters for MMP-1 hydrolysis of fTHP-3 were k cat = 0.080 s-1 and K M = 61.2 μM. Subsequent investigations showed fTHP-3 hydrolysis by MMP-2, MMP-3, MMP-13, a C-terminal domain-deleted MMP-1 [MMP-1(Δ243 - 450)], and a C-terminal domain-deleted MMP-3 [MMP-3(Δ248 - 460)]. The order of k cat/K M values was MMP-13 > MMP-1 ∼ MMP-1(Δ243 - 450) ∼ MMP-2 ≫ MMP-3 ∼ MMP-3(Δ248 - 460). Studies on the effect of temperature on fTHP-3 and fSSP-3 hydrolysis by MMP-1 showed that the activation energies between these two substrates differed by 3.4-fold, similar to the difference in activation energies for MMP-1 hydrolysis of type I collagen and gelatin. This indicates that fluorogenic triple-helical substrates mimic the behavior of the native collagen substrate and may be useful for the investigation of collagenase triple-helical activity.