Reaction mechanism of glutamate carboxypeptidase II revealed by mutagenesis, X-ray crystallography, and computational methods.
Reaction mechanism of glutamate carboxypeptidase II revealed by mutagenesis, X-ray crystallography, and computational methods.
复制标题
DOI：
10.1021/bi900220s
复制
发表时间：
2009-05-19
期刊：
影响因子：
2.9
通讯作者：
Lubkowski J
中科院分区：
文献类型：
--
作者：
Klusák V;Barinka C;Plechanovová A;Mlcochová P;Konvalinka J;Rulísek L;Lubkowski J
谷氨酸羧肽酶 II (GCPII, EC 3.4.17.21) 是一种锌依赖性外肽酶，是神经退行性疾病和前列腺癌的重要治疗靶点。 N-乙酰基-L-天冬氨酰-L-谷氨酸 (N-Ac-Asp-Glu) 是 GCPII 的天然二肽底物，其水解与哺乳动物神经系统内的细胞信号传导密切相关，但该反应的确切机制尚未确定。为了详细研究 GCPII 的肽水解作用，我们构建了人 GCPII 的突变体 [GCPII(E424A)]，其中假定的质子穿梭残基 Glu424 被丙氨酸取代。使用 N-Ac-Asp-Glu 作为底物的 GCPII(E424A) 动力学分析显示催化活性完全丧失，表明 Glu424 直接参与肽水解。此外，我们以 1.70 Å 的分辨率测定了 GCPII(E424A) 与 N-Ac-Asp-Glu 复合物的晶体结构。 GCPII(E424A) 结合腔中完整底物的存在证实了我们的动力学数据，并允许对 GCPII/N-Ac-Asp-Glu 相互作用进行详细分析。实验数据得到量子力学/分子力学组合计算 (QM/MM) 的补充，使我们能够表征过渡态，包括相关的反应势垒，并提供有关 GCPII 反应机制的详细信息。反应势垒的最佳估计计算为 ΔG‡ ≈ 22((±5) kcal · mol−1，这与实验观察到的反应速率常数 (kcat ≈ 1 s−1) 非常吻合。综合起来，我们的结果在原子水平上提供了这种非常有趣的酶的反应机制的详细且一致的图像。
Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a zinc-dependent exopeptidase and an important therapeutic target for neurodegeneration and prostate cancer. The hydrolysis of N-acetyl-l-aspartyl-l-glutamate (N-Ac-Asp-Glu), the natural dipeptidic substrate of the GCPII, is intimately involved in cellular signaling within the mammalian nervous system, but the exact mechanism of this reaction has not yet been determined. To investigate peptide hydrolysis by GCPII in detail, we constructed a mutant of human GCPII [GCPII(E424A)], in which Glu424, a putative proton shuttle residue, is substituted with alanine. Kinetic analysis of GCPII(E424A) using N-Ac-Asp-Glu as substrate revealed a complete loss of catalytic activity, suggesting the direct involvement of Glu424 in peptide hydrolysis. Additionally we determined the crystal structure of GCPII(E424A) in complex with N-Ac-Asp-Glu at 1.70 Å resolution. The presence of the intact substrate in the GCPII(E424A) binding cavity substantiates our kinetic data and allows a detailed analysis of GCPII/N-Ac-Asp-Glu interactions. The experimental data are complemented by the combined quantum mechanics/molecular mechanics calculations (QM/MM) which enabled us to characterize the transition states, including the associated reaction barriers, and provided detailed information concerning the GCPII reaction mechanism. The best estimate of the reaction barrier was calculated to be ΔG‡ ≈ 22((±5) kcal · mol−1, which is in a good agreement with the experimentally observed reaction rate constant (kcat ≈ 1 s−1). Combined together, our results provide a detailed and consistent picture of the reaction mechanism of this highly interesting enzyme at the a tomic level.
登录
查看更多内容
DOI：
10.1107/s0907444900002420
发表时间：
2000-05-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Gilboa, R;Greenblatt, HM;Shoham, G
通讯作者：
Shoham, G
影响因子：
2.6
作者：
Kinoshita, Y;Kuratsukuri, K;Haas, GP
通讯作者：
Haas, GP
影响因子：
4.4
作者：
BECKE, AD
通讯作者：
BECKE, AD
影响因子：
5.6
作者：
Greenblatt, HM;Almog, O;Shoham, G
通讯作者：
Shoham, G
影响因子：
3.5
作者：
Fundoiano-Hershcovitz, Y;Rabinovitch, L;Shoham, Y
通讯作者：
Shoham, Y