Berg R A, Kishida Y, Sakakibara S, Prockop D J
Biochemistry. 1977 Apr 19;16(8):1615-21. doi: 10.1021/bi00627a014.
Previous studies with 14C-labeled synthetic peptides demonstrated that prolyl hydroxylase, which synthesizes the hydroxyproline in collagen, preferentially hydroxylates the fourth triplet from the NH-terminal end of the peptide (Pro-Pro-Gly)5. In the experiments reported here, the prolyl hydroxylase reaction was investigated further by preparing chemically modified derivatives of (Pro-Pro-Gly)5 and by synthesizing 14C-labeled preparations of (Pro-Pro-Gly)10. Essentially, the same kcat value was found for the hydroxylation of (Pro-Pro-Gly)5, N-acetyl-(Pro-Pro-Gly)5, (Pro-Pro-Gly)5 methyl ester, (Pro-Pro-Gly)10, and for larger polypeptide substrates of the enzyme. It appeared therefore that preferential hydroxylation of specific triplets in peptides of the structure (Pro-Pro-Gly)n cannot be explained by differences in the kinetic constants for individual triplets. Hydroxylation of 14C-labeled preparations of (Pro-Pro-Gly)10 demonstrated that the ninth triplet was preferentially hydroxylated over any other triplet. The results were best explained by the hypothesis that prolyl hydroxylase has an asymmetric active site in which binding subsites are located adjacent to but not symmetrical with the catalytic subsite.
先前使用14C标记的合成肽进行的研究表明，在胶原蛋白中合成羟脯氨酸的脯氨酰羟化酶优先羟基化肽（脯氨酸-脯氨酸-甘氨酸）5氨基末端的第四个三联体。在本文报道的实验中，通过制备（脯氨酸-脯氨酸-甘氨酸）5的化学修饰衍生物和合成（脯氨酸-脯氨酸-甘氨酸）10的14C标记制剂，对脯氨酰羟化酶反应进行了进一步研究。基本上，发现（脯氨酸-脯氨酸-甘氨酸）5、N-乙酰基-（脯氨酸-脯氨酸-甘氨酸）5、（脯氨酸-脯氨酸-甘氨酸）5甲酯、（脯氨酸-脯氨酸-甘氨酸）10以及该酶的更大多肽底物的羟基化具有相同的催化常数（kcat）值。因此，似乎结构为（脯氨酸-脯氨酸-甘氨酸）n的肽中特定三联体的优先羟基化不能用单个三联体动力学常数的差异来解释。（脯氨酸-脯氨酸-甘氨酸）10的14C标记制剂的羟基化表明，第九个三联体比任何其他三联体更优先被羟基化。这些结果最好用脯氨酰羟化酶具有不对称活性位点的假说来解释，在该位点中结合亚位点位于催化亚位点附近但不对称。
