Kinetic analysis of the mechanism of interaction of full-length TIMP-2 and gelatinase A: evidence for the existence of a low-affinity intermediate.
We have undertaken a detailed analysis of the mechanism of inhibition of matrix metalloproteinase-2 (gelatinase A) by tissue inhibitor of metalloproteinase-2 (TIMP-2). Quenched fluorescent substrates have been used to analyze the rate of inhibition of gelatinase A by TIMP-2 over a wide range of TIMP-2 concentrations. When the values of the observed rate constant for the inhibition are plotted against TIMP-2 concentration, saturation is observed at high concentrations, providing evidence for formation of an intermediate in the pathway. Rate constants for the formation and dissociation of the intermediate are 5.9 x 10(6) M-1 s-1 and 6.3 s-1 respectively, giving a Ki for the initial step of approximately 1 microM. The rate constant for the association of the final complex is 33 s-1. By studying the dissociation of 125I-labeled TIMP-2 from a gelatinase A-TIMP-2 complex using ligand exchange experiments, we obtained a rate constant for the dissociation of the final stable complex of 2 x 10(-)8 s-1. This gives a value for the overall dissociation constant of approximately 0.6 fM.
School of Biological Sciences, University of East Anglia, Norwich, UK. m.hutton@uea.ac.uk
M
M
Hutton
F
F
Willenbrock
K
K
Brocklehurst
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G
Murphy
D001667:Binding, Competitive; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism; D018093:Gelatinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D020778:Matrix Metalloproteinase 2; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000737:chemistry / Q000378:metabolism*
C095851:7-methoxycoumarin-4-yl-acetyl-prolyl-leucyl-glycyl-leucyl-(3-(2,4-dinitrophenyl)-2,3-diaminopropanoyl)-alanyl-argininamide; D005456:Fluorescent Dyes; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D009842:Oligopeptides; D019716:Tissue Inhibitor of Metalloproteinase-2; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2