The activity of the tissue inhibitors of metalloproteinases is regulated by C-terminal domain interactions: a kinetic analysis of the inhibition of gelatinase A.
The cloning and expression of the full-length tissue inhibitor of metalloproteinase 2 (TIMP-2), delta 187-194TIMP-2, and delta 128-194TIMP-2 and the purification of these inhibitors and a cleaved version of TIMP-2 lacking nine C-terminal amino acids (delta 186-194TIMP-2) are described. The mechanism of inhibition of gelatinase A by the TIMPs was investigated by comparing the kinetics of association of TIMP-1, TIMP-2, the C-terminal deletions, and the mutants of both TIMPs which consisted of the N-terminal domain only. The full-length TIMPs inhibited gelatinase A rapidly with association constants of 3.2 x 10(6) M-1 s-1 for TIMP-1 and 2.1 x 10(7) M-1 s-1 for TIMP-2 at I = 0.2. The C-terminal peptide of TIMP-2 is proposed to exist as an exposed "tail" responsible for binding to progelatinase A and for increasing the rate of inhibition of active gelatinase A through electrostatic interactions with the C-terminal domain of the enzyme. The C-terminal domains of both TIMP-1 and TIMP-2 participate in low-affinity interactions with the C-terminal domain of gelatinase A which increase the rate of association by a factor of about 100 in both cases.
Willenbrock
F
F
Department of Biochemistry, Queen Mary and Westfield College, University of London, U.K.
Crabbe
T
T
Slocombe
P M
PM
Sutton
C W
CW
Docherty
A J
AJ
Cockett
M I
MI
O'Shea
M
M
Brocklehurst
K
K
Phillips
I R
IR
Murphy
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008433:Mathematics; D020778:Matrix Metalloproteinase 2; D061965:Matrix Metalloproteinase Inhibitors; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D009101:Multiple Myeloma; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping; D019714:Tissue Inhibitor of Metalloproteinases; D014407:Tumor Cells, Cultured
D006023:Glycoproteins; D061965:Matrix Metalloproteinase Inhibitors; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2