A SOLUBLE METALLOENDOPEPTIDASE FROM RAT-BRAIN - PURIFICATION OF THE ENZYME AND DETERMINATION OF SPECIFICITY WITH SYNTHETIC AND NATURAL PEPTIDES
A SOLUBLE METALLOENDOPEPTIDASE FROM RAT-BRAIN - PURIFICATION OF THE ENZYME AND DETERMINATION OF SPECIFICITY WITH SYNTHETIC AND NATURAL PEPTIDES
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DOI：
10.1111/j.1432-1033.1983.tb07620.x
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发表时间：
1983-01-01
期刊：
影响因子：
--
通讯作者：
CHU, TG
中科院分区：
文献类型：
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作者：
ORLOWSKI, M;MICHAUD, C;CHU, TG
从脑匀浆的可溶部分中纯化出一种在中性pH下活性最佳的金属内肽酶。酶（MW .apprx.）67,000)被EDTA和邻菲罗啉等金属螯合剂强烈抑制。edta处理后的酶可以被几种二价金属离子（包括Zn2+、Co2+和Mn2+）重新激活。用一系列模型合成底物研究了该酶的特异性和动力学参数。该酶优先切割肽键，其中羰基是由芳香氨基酸残基在P1位置贡献的。芳香残基位于P3”和P1位置或同时位于P3”和P1和P2位置时，Km值最低，kcat /Km比值最高。在P1位置有精氨酸残基的底物得到较低的kcat/Km比，在P1位置有甘氨酸或天冬氨酸残基的底物得到更低的kcat/Km比。在P1或P1“位置引入d -氨基酸残基，使底物完全耐水解。特异性研究表明，金属内肽酶的活性位点可以容纳至少5个氨基酸残基，其中2个残基结合在水解键的n端，3个结合在c端。一些生物活性肽被酶在与模型合成底物研究推断的特异性一致的位点上切割。
A metalloendopeptidase, optimally active at a neutral pH, was purified from the soluble fraction of brain homogenates. The enzyme (MW .apprx. 67,000) is strongly inhibited by metal chelators such as EDTA and o-phenanthroline. An EDTA-treated enzyme can be reactivated by several divalent metal ions including Zn2+, Co2+ and Mn2+. The specificity and kinetic parameters of the enzyme were studied with a series of model synthetic substrates. The enzyme preferentially cleaves peptide bonds in which the carbonyl group is contributed by an aromatic amino acid residue in the P1 position. The lowest Km values and the highest kcat[catalytic rate constant]/Km ratios were obtained with substrates having aromatic residues in the P3'' and P1 position or in the P3'' and both the P1 and P2 positions. Lower kcat/Km ratios were obtained with substrates having arginine residues in position P1, and even lower values with those substrates having a glycine or aspartyl residue in this position. Introduction of a D-amino acid residue in either position P1 or P1'' renders the substrate totally resistant to hydrolysis. The specificity studies suggest that the active site of the metalloendopeptidase can accommodate at least 5 amino acid residues, with 2 of those residues binding on the N-terminal side and 3 binding on the C-terminal side of the hydrolyzed bond. Several biologically active peptides are cleaved by the enzyme at sites consistent with the specificity deduced from studies with model synthetic substrates.