Knight C G, Dando P M, Barrett A J
Department of Biochemistry, Strangeways Research Laboratory, Worts Causeway, Cambridge, U.K.
Biochem J. 1995 May 15;308 ( Pt 1)(Pt 1):145-50. doi: 10.1042/bj3080145.
The substrate-size specificity of human thimet oligopeptidase (EC 3.4.24.15) was investigated with oligomers of glycyl-prolyl-leucine (GPL)n where n = 2, 3, 4 and 5. These peptides were cleaved only at Leu-Gly bonds to give GPL as the single final product. Hydrolysis was most rapid with (GPL)3 and slowest with (GPL)5. The more water-soluble oligomers of Gly-Hyp-Leu showed the same trend. (Gly-Hyp-Leu)6 was not hydrolysed, consistent with the previous finding that substrates larger than 17 amino acids are not cleaved by thimet oligopeptidase. The cleavage of (GPL)3 to GPL fitted a sequential first-order model. First-order kinetics were unexpected as the initial substrate concentration was greater than Km. The anomaly was also seen during the cleavage of bradykinin and neurotensin, and in these cases first-order behaviour was due to potent competitive inhibition by the C-terminal product. The sequential mechanism for (GPL)3 breakdown by thimet oligopeptidase does not discriminate between initial cleavages towards the N- or C-terminus. As isoleucine is an unfavourable residue in P1, substrates were made in which selected leucine residues were replaced by isoleucine. GPL--GPI--GPL (where--represents the bond between the tripeptide units) was resistant to hydrolysis and GPI--GPL--GPL was cleaved only at the -Leu-Gly- bond. Experiments with isoleucine-containing analogues of (Gly-Hyp-Leu)4 showed that thimet oligopeptidase preferred to cleave these peptides near the C-terminus.
利用甘氨酰-脯氨酰-亮氨酸（GPL）n（n = 2、3、4和5）的寡聚体研究了人硫醇寡肽酶（EC 3.4.24.15）的底物大小特异性。这些肽仅在Leu-Gly键处裂解，生成GPL作为唯一的最终产物。水解速度以（GPL）3最快，以（GPL）5最慢。甘氨酰-羟脯氨酰-亮氨酸的水溶性更高的寡聚体也显示出相同的趋势。（甘氨酰-羟脯氨酰-亮氨酸）6未被水解,这与之前的发现一致，即大于17个氨基酸的底物不会被硫醇寡肽酶裂解。（GPL）3裂解为GPL符合顺序一级模型。由于初始底物浓度大于Km，出现一级动力学是出乎意料的。在缓激肽和神经降压素的裂解过程中也观察到了这种异常情况，在这些情况下，一级行为是由于C末端产物的强效竞争性抑制作用。硫醇寡肽酶对（GPL）3进行分解的顺序机制无法区分对N末端或C末端的初始裂解。由于异亮氨酸在P1中是一个不利的残基，因此制备了一些底物，其中选定的亮氨酸残基被异亮氨酸取代。GPL--GPI--GPL（其中--代表三肽单元之间的键）对水解具有抗性，而GPI--GPL--GPL仅在-Leu-Gly-键处裂解。对（甘氨酰-羟脯氨酰-亮氨酸）4的含异亮氨酸类似物的实验表明，硫醇寡肽酶更倾向于在这些肽的C末端附近进行裂解。
