Willenbrock F, Crabbe T, Slocombe P M, Sutton C W, Docherty A J, Cockett M I, O'Shea M, Brocklehurst K, Phillips I R, Murphy G
Department of Biochemistry, Queen Mary and Westfield College, University of London, U.K.
Biochemistry. 1993 Apr 27;32(16):4330-7. doi: 10.1021/bi00067a023.
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.
本文描述了金属蛋白酶组织抑制剂2（TIMP-2）全长、δ187 - 194TIMP-2、δ128 - 194TIMP-2的克隆与表达，以及这些抑制剂和缺失九个C末端氨基酸的TIMP-2裂解版本（δ186 - 194TIMP-2）的纯化。通过比较TIMP-1、TIMP-2、C末端缺失片段以及仅由N末端结构域组成的两种TIMP突变体的结合动力学，研究了TIMP对明胶酶A的抑制机制。在离子强度I = 0.2时，全长TIMP对明胶酶A的抑制作用迅速，TIMP-1的结合常数为3.2×10⁶ M⁻¹ s⁻¹，TIMP-2的结合常数为2.1×10⁷ M⁻¹ s⁻¹。TIMP-2的C末端肽被认为以暴露的“尾巴”形式存在，负责与前明胶酶A结合，并通过与酶的C末端结构域的静电相互作用提高对活性明胶酶A的抑制速率。TIMP-1和TIMP-2的C末端结构域均参与与明胶酶A的C末端结构域的低亲和力相互作用，在两种情况下，这种相互作用都使结合速率提高了约100倍。
