A structure-function study of a proton transport pathway in the gamma-class carbonic anhydrase from Methanosarcina thermophila.
A structure-function study of a proton transport pathway in the gamma-class carbonic anhydrase from Methanosarcina thermophila.
复制标题
嗜热甲烷八叠球菌γ类碳酸酐酶质子转运途径的结构-功能研究。
DOI：
10.1021/bi0001877
复制
发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Ferry,JG
中科院分区：
文献类型：
--
作者：
Tripp,BC;Ferry,JG
利用定点突变和化学拯救技术对嗜热甲烷八叠球菌（Methanosarcina thermophila，Cam）γ-类碳酸酐酶的4个谷氨酸残基进行了研究。丙氨酸取代表明，外部环残基，Glu 84，和内部活性位点残基，Glu 62，都是重要的CO2水合活性。另外两个外环残基，Glu 88和Glu 89，对酶功能不太重要。两种E84 D和-H变体在pH 7.5 MOPS缓冲液中相对于野生型活性表现出显著的活性，表明原始谷氨酸残基可以被具有相似pKa值的其他可电离残基取代。E84 A、-C、-K、-Q、-S和-Y变体在pH 7.5的MOPS缓冲液中表现出大的inkcat值降低，但仅表现出小的inkcat/Km变化。这六种变异体都被pH 7.5的咪唑缓冲液化学拯救，表观kcat增加了23 - 46倍。这些结果与Glu 84作为质子穿梭残基起作用一致。在pH7.5的MOPS缓冲液中，E62 D变体显示出相对于野生型的3倍降低的inkcat和2倍降低的inkcat/Km，而其它取代（E62 A、-C、-H、-Q、-T和-Y）导致kcat和kcat/Km的更大的降低。咪唑对大多数Glu 62变异体的kcat值无显著增加作用，而对kcat/Km值有轻微降低作用。这些结果表明，在位置62的羧酸基团的主要偏好，并支持建议的催化作用，残基谷氨酸62在CO2水合步骤，但不明确建立其在质子传输步骤的作用。
Four glutamate residues in the prototypic γ-class carbonic anhydrase fromMethanosarcina thermophila(Cam) were characterized by site-directed mutagenesis and chemical rescue studies. Alanine substitution indicated that an external loop residue, Glu 84, and an internal active site residue, Glu 62, are both important for CO2hydration activity. Two other external loop residues, Glu 88 and Glu 89, are less important for enzyme function. The two E84D and -H variants exhibited significant activity relative to wild-type activity in pH 7.5 MOPS buffer, suggesting that the original glutamate residue could be substituted with other ionizable residues with similar pKavalues. The E84A, -C, -K, -Q, -S, and -Y variants exhibited large decreases inkcatvalues in pH 7.5 MOPS buffer, but only exhibited small changes inkcat/Km. These same six variants were all chemically rescued by pH 7.5 imidazole buffer, with 23−46-fold increases in the apparentkcat. These results are consistent with Glu 84 functioning as a proton shuttle residue. The E62D variant exhibited a 3-fold decrease inkcatand a 2-fold decrease inkcat/Kmrelative to those of the wild type in pH 7.5 MOPS buffer, while other substitutions (E62A, -C, -H, -Q, -T, and -Y) resulted in much larger decreases in bothkcatandkcat/Km. Imidazole did not significantly increase thekcatvalues and slightly decreased thekcat/Kmvalues of most of the Glu 62 variants. These results indicate a primary preference for a carboxylate group at position 62, and support a proposed catalytic role for residue Glu 62 in the CO2hydration step, but do not definitively establish its role in the proton transport step.