BIOCHEMICAL-CHARACTERIZATION OF MATRILYSIN - ACTIVATION CONFORMS TO THE STEPWISE MECHANISMS PROPOSED FOR OTHER MATRIX METALLOPROTEINASES
BIOCHEMICAL-CHARACTERIZATION OF MATRILYSIN - ACTIVATION CONFORMS TO THE STEPWISE MECHANISMS PROPOSED FOR OTHER MATRIX METALLOPROTEINASES
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DOI：
10.1021/bi00151a017
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发表时间：
1992-09-15
期刊：
影响因子：
2.9
通讯作者：
DOCHERTY, AJP
中科院分区：
文献类型：
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作者：
CRABBE, T;WILLENBROCK, F;DOCHERTY, AJP
从转染的小鼠骨髓瘤细胞条件培养液中纯化了基质溶素的潜在前体(EC 3.4.24.23；PUMP)，发现每个分子含有一个锌原子，这是催化活性所必需的。(4-氨基苯基)醋酸汞、胰酶和高温孵育(热激活)均可使原溶血酶活化到相同的比活力。在最适pH为6.5，pK(A)为4.6和8.6 5的条件下，活性基质溶酶在甘氨酸亮氨酸键上对荧光底物2，4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2的最大水解量k(CAT)/K(M)为1.3×10(4)M-1 S-1。金属蛋白酶组织抑制物-1以1：1的化学计量比相互作用抑制活性。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法结合N-末端测序分析表明，与所有其他类似研究的基质金属蛋白酶一样，原溶血素的激活伴随着从N-末端逐步去除M(R)9000前肽的过程。产生的中间体取决于所使用的激活方式，但在所有研究的情况下，激活以E77-Y78处的自催化裂解终止，最终产生M(R)19000活性基质溶酶。从各种中间体的稳定性分析中，我们认为L13-K33序列在保护E77-Y78位点不被自动催化切割从而保持酶原的潜伏期方面特别重要。
The latent precursor of matrilysin (EC 3.4.24.23; punctuated metalloproteinase (PUMP)) was purified from transfected mouse myeloma cell conditioned medium and was found to contain one zinc atom per molecule which was essential for catalytic activity. Promatrilysin could be activated to the same specific activity by (4-aminophenyl)mercuric acetate, trypsin, and incubation at elevated temperatures (heat activation). Active matrilysin hydrolyzed the fluorescent substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond with a maximum value for k(cat)/K(m) of 1.3 x 10(4) M-1 s-1 at the pH optimum of 6.5 and pK(a) values of 4.60 and 8.65. Activity is inhibited by the tissue inhibitor of metalloproteinases-1 in a 1:1 stoichiometric interaction. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with N-terminal sequencing revealed that, as with all other matrix metalloproteinases similarly studied, promatrilysin activation was accompanied by the stepwise proteolytic removal of an M(r) 9000 propeptide from the N-terminus. The intermediates generated were dependent on the mode of activation used but, in all cases studied, activation terminated with an autocatalytic cleavage at E77-Y78 to yield the final M(r) 19 000 active matrilysin. From an analysis of the stability of the various intermediates, we propose that the sequence L13-K33 is particularly important in protecting the E77-Y78 site from autocatalytic cleavage, thereby maintaining the latency of the proenzyme.