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.
The cysteine proteinase superfamily is a source of natural structural variants of value in the investigation of mechanism. It has long been considered axiomatic that catalytic competence of these enzymes mirrors the generation of the ubiquitous catalytic site imidazolium-thiolate ion pair. We here report definitive evidence from kinetic studies supported by electrostatic potential calculations, however, that at least for some of these enzymes the ion pair state which provides the nucleophilic and acid-base chemistry is essentially fully developed at low pH where the enzymes are inactive. Catalytic competence requires an additional protonic dissociation with a common pKa value close to 4 possibly from the Glu50 cluster to control ion pair geometry. The pH dependence of the second-order rate constant (k) for the reactions of the catalytic site thiol groups with 4,4'-dipyrimidyl disulfide is shown to provide the pKa values for the formation and deprotonation of the (Cys)-S-/(His)-Im+H ion pair state. Analogous study of the reactions with 2,2'-dipyridyl disulfide reveals other kinetically influential ionizations, and all of these pKa values are compared with those observed in the pH dependence of kcat/Km for the catalyzed hydrolysis of N-acetylphenylalanylglycine 4-nitroanilide. The discrepancy between the pKa value for ion pair formation and the common pKa value close to 4 related to generation of catalytic activity is particularly marked for ficin (pKa 2.49 +/- 0.02) and caricain (pKa 2.88 +/- 0.02) but exists also for papain (pKa 3.32 +/- 0.01).
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