ON SIZE OF ACTIVE SITE IN PROTEASES .2. CARBOXYPEPTIDASE-A
ON SIZE OF ACTIVE SITE IN PROTEASES .2. CARBOXYPEPTIDASE-A
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DOI：
10.1016/0006-291x(67)90299-9
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发表时间：
1967-01-01
影响因子：
3.1
通讯作者：
BERGER, A
中科院分区：
文献类型：
--
作者：
ABRAMOWITZ, N;SCHECHTER, I;BERGER, A
通过研究L-丙氨酸、D-丙氨酸和L-苯丙氨酸肽以及它们的N-苄氧基羰基、N-乙酰基、N-苯丙酰基和N-甲氧羰基衍生物的水解动力学，研究了羧肽酶-A（来自牛胰腺）活性部位的大小。从各种动力学参数（[image]m，kcat）的比较可以得出结论，该酶的活性位点延伸超过约18 A。结合区可分为5个“亚位点”。每一个容纳底物的1个氨基酸残基（或封闭基团）。通过比较在相同位置含有甲基或苄基侧链的底物对的m值，结果表明，结合区域作为一个整体对芳香族残基具有更大的亲和力。用D-残基替换L-残基会降低kcat值，而不是[image]m。此外，还发现对位于S3亚位点的乙酰基有明显的亲和力，与乙酰基或苯丙酰基相比，占据该亚位点的甲氧羰基或苄氧羰基的Km值分别增加了5倍。亚位点S2或S4中的甲氧羰基或苄氧羰基没有这种作用。
The size of the active site of carboxypeptidase-A [from bovine pancreas] was investigated by studying the kinetics of hydrolysis of peptides of L-alanine, D-alanine and L-phenylalanine, as well as of a number of their N-benzyloxy-carbonyl, N-acetyl, N-phenylproprionyl and N-methyloxycarbonyl derivatives. From a comparison of the various kinetic parameters ([image]m, kcat) it was concluded that the active site of this enzyme extends over about 18 A. The binding area can be divided into 5 "subsites". each accommodating 1 amino acid residue (or blocking group) of the substrate. By comparing [image]m values of pairs of substrates containing either a methyl or a benzyl side-chain in equivalent positions, it was shown that the binding area as a whole has a larger affinity towards the aromatic residues. Substitution of a D-residue for an L-resi-due reduced kcat values rather than [image]m. In addition a remarkable affinity for the urethane-grouping located specifically at subsite S3 was found. A methyloxycarbonyl or benzyloxy-carbonyl group occupying this subsite caused a 5-fold increase in Km as compared with an acetyl or phenylproprionyl group, respectively. Methyloxycarbonyl or benzyloxycarbonyl groups in subsites S2 or S4 showed no such effect.