CARBOXYPEPTIDASE-A CATALYZED-HYDROLYSIS OF THIOPEPTIDE AND THIONESTER ANALOGS OF SPECIFIC SUBSTRATES - AN EFFECT ON KCAT FOR PEPTIDE, BUT NOT ESTER, SUBSTRATES
CARBOXYPEPTIDASE-A CATALYZED-HYDROLYSIS OF THIOPEPTIDE AND THIONESTER ANALOGS OF SPECIFIC SUBSTRATES - AN EFFECT ON KCAT FOR PEPTIDE, BUT NOT ESTER, SUBSTRATES
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DOI：
10.1021/ja00383a038
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发表时间：
1982-01-01
影响因子：
15
通讯作者：
NASHED, NT
中科院分区：
文献类型：
--
作者：
CAMPBELL, P;NASHED, NT
羧肽酶A [牛胰腺]分别催化N-（N-马尿酰硫代甘氨酰）-3-苯基-L-丙氨酸、O-（N-马尿酰甘氨酰）-3-苯基-L-乳酸和O-（N-马尿酰硫代甘氨酰）-3-苯基-L-乳酸（其特异性三肽底物N-（N-马尿酰甘氨酰）-3-苯基-L-丙氨酸的硫肽、酯和硫酮酯类似物）的水解。酯和硫酮酯底物的kcat和kcat/Km均相等，反映了这2种化合物的非酶反应性相等。然而，硫代肽的kcat/Km仅为其肽对应物的kcat/Km的0.0009。这种差异不是由于酰胺和硫代酰胺之间的任何固有反应性差异造成的，而是由于硫代肽和肽同样良好地结合而存在的。kcat-和kcat/Km-pH曲线的控制pKa与先前观察到的特定酯和肽底物的pKa相匹配。由于旋转硫代酰胺键约3千卡mol-1比旋转一个肽键更困难，这些数据支持一种机制，涉及速率决定键旋转肽酶，但不酯酶，活性。
Carboxypeptidase A [bovine pancreas] catalyzes the hydrolysis of N-(N-hippurylthioglycyl)-3-phenyl-L-alanine, O-(N-hippurylglycyl)-3-phenyl-L-lactic acid, and O-(N-hippurylthioglycyl)-3-phenyl-L-lactic acid, respectively, the thiopeptide, ester and thionester analogs of its specific tripeptide substrate N-(N-hippurylglycyl)-3-phenyl-L-alanine. Both kcat and kcat/Km are equal for the ester and thionester substrates, reflecting the equal nonenzymic reactivities for these 2 compounds. However, kcat/Km for the thiopeptide is only 0.0009 as large as that for its peptide counterpart. This difference, which cannot be due to any inherent reactivity differences between amides and thioamides, lies in kcat, since thiopeptide and peptide bind equally well. The controlling pKa of the kcat- and kcat/Km-pH profiles match those previously observed for specific ester and peptide substrates. Since rotation about the thioamide bond is about 3 kcal mol-1 more difficult than rotation about a peptide bond, these data support a mechanism involving rate-determining bond rotation in peptidase, but not esterase, activity.