Role of the conserved histidine and aspartic acid residues in activity and stabilization of human gelatinase B: an example of matrix metalloproteinases.
Role of the conserved histidine and aspartic acid residues in activity and stabilization of human gelatinase B: an example of matrix metalloproteinases.
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保守的组氨酸和天冬氨酸残基在人明胶酶 B 的活性和稳定性中的作用：基质金属蛋白酶的一个例子。
DOI：
10.1007/bf01886879
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Mainardi,CL
中科院分区：
文献类型：
--
作者：
Pourmotabbed,T;Aelion,JA;Tyrrell,D;Hasty,KA;Bu,CH;Mainardi,CL
明胶酶B（MMP-9）是基质金属蛋白酶家族的成员，是已知在肿瘤细胞侵袭和关节炎中软骨破坏中起作用的锌和钙依赖性内肽酶。其保守序列为400 His-（X）3-His-（X）28-Asp-Asp-（X）2- 436 Gly，其功能尚待进一步研究。保守的Asp-432和Asp-433残基被单独替换为Gly;这些取代使酶的明胶分解活性分别降低至23%和0%。用Glu替代Asp-433，酶的明胶分解活性降低93%，对Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH 2底物的蛋白水解活性降低79%。野生型和D432 G和D433 E突变酶对合成底物具有相似的Km值，对竞争性抑制剂GM 6001具有相似的Ki值。然而，D432 G和D433 E突变酶的kcat/Km值减少了1.44倍，它们的KaCa值分别增加了4倍和6倍。His-400在酶活性中的重要性通过用Ala和Phe替换该残基来评估。H400 A和H400 F突变体均对明胶底物无活性。这些数据表明，Asp-432、Asp-433和His-400残基对明胶酶B的活性很重要。His-400可能与His-197类似，在间质胶原酶（MMP-7）中作为锌结合配体，Asp-432和Asp-433残基可能参与酶活性位点的稳定。His-400和Asp-433残基在MMP家族的所有成员中是保守的。因此，我们的研究结果与整个群体相关。
Gelatinase B (MMP-9), a member of the matrix metalloproteinase family, is a zinc- and calcium-dependent endopeptidase that is known to play a role in tumor cell invasion and in destruction of cartilage in arthritis. It contains a conserved sequence400His-(X)3-His-(X)28-Asp-Asp-(X)2-436Gly, the function of which is under investigation. The conserved Asp-432 and Asp-433 residues were individually replaced with Gly; these substitutions reduced the gelatinolytic activity of the enzyme to 23% and 0%, respectively. Replacing Asp-433 with Glu, however, decreased the gelatinolytic activity of the enzyme by 93% and proteolytic activity of the enzyme for the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2substrate by 79%. The wild-type and D432G and D433E mutant enzymes had similarKmvalues for the synthetic substrate and similarKivalues for the competitive inhibitor, GM6001. Thekcat/Kmvalues for D432G and D433E mutant enzymes, however, were reduced by a factor of ∼4 and their KaCavalues were increased by four- and sixfold, respectively. The significance of His-400 in the activity of the enzyme was assessed by replacing this residue with Ala and Phe. Both H400A and H400F mutants were inactive toward gelatin substrate. These data demonstrate that Asp-432, Asp-433, and His-400 residues are important for the activity of gelatinase B. His-400 may act as a zinc-binding ligand similar to the His-197 in interstitial collagenase (MMP-7) and Asp-432 and Asp-433 residues are probably involved in stabilization of the active site of the enzyme. The His-400 and Asp-433 residues are conserved in all members of the MMP family. Therefore, our results are relevant to this group as a whole.