Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli.
Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli.
复制标题
DOI：
10.1021/bi9003759
复制
发表时间：
2009-05-26
期刊：
影响因子：
2.9
通讯作者：
Cleland, W. Wallace
中科院分区：
文献类型：
--
作者：
Zeczycki, Tonya N.;St Maurice, Martin;Jitrapakdee, Sarawut;Wallace, John C.;Attwood, Paul V.;Cleland, W. Wallace
研究了草酰丙酮酸羧化酶羧基转移酶活性部位的突变对正向反应生成草酰乙酸酯、反向反应生成镁三磷酸腺苷、草甲酸酯诱导的脱羧基、氨基甲酰磷酸对镁ADP的磷酸化以及重碳酸盐依赖的ATPase反应的影响。对这些突变体的进一步研究考察了丙酮酸和草酸对生物素羧化酶结构域反应的影响。从这些诱变研究中，指定了与催化相关的活性部位残基的假定作用，并对羧基转移酶结构域的机制进行了更准确的描述。T882a突变体对涉及羧基转移酶结构域的反应没有催化活性，但令人惊讶的是，与野生型相比，T882a突变体对ADP磷酸化和重碳酸盐依赖的ATPase反应的活性分别增加了7倍和3.5倍。此外，草酸酯和丙酮酸对T882A催化的BC结构域反应的部分抑制进一步支持了Thr882在羧基转移酶结构域生物素和丙酮酸之间的质子转移中的关键作用。其催化机制似乎涉及到羧基生物素的脱羧化和生成的生物素烯醇化产物将质子从Thr882中移除，同时将质子从丙酮酸盐协同或随后转移到Thr882。生成的烯醇式丙酮酸然后与二氧化碳反应生成草酰乙酸酯并完成反应。
The effects of mutations in the active site of the carboxyl transferase domain of R. etli pyruvate carboxylase have been determined for the forward reaction to form oxaloacetate, the reverse reaction to form MgATP, the oxamate-induced decarboxylation of oxaloacetate, the phosphorylation of MgADP by carbamoyl phosphate and the bicarbonate-dependent ATPase reaction. Additional studies with these mutants examined the effect of pyruvate and oxamate on the reactions of the biotin carboxylase domain. From these mutagenic studies, putative roles for catalytically relevant active site residues were assigned and a more accurate description of the mechanism of the carboxyl transferase domain is presented. The T882A mutant showed no catalytic activity for reactions involving the carboxyl transferase domain, but surprisingly showed a 7- and 3.5-fold increase in activity, as compared to the wild-type enzyme, for the ADP phosphorylation and bicarbonate-dependent ATPase reactions, respectively. Furthermore, the partial inhibition of the T882A catalyzed BC domain reactions by oxamate and pyruvate further supports the critical role of Thr882 in the proton transfer between biotin and pyruvate in the carboxyl transferase domain. The catalytic mechanism appears to involve the decarboxylation of carboxybiotin and proton removal from Thr882 by the resulting biotin enolate with either a concerted or subsequent transfer of a proton from pyruvate to Thr882. The resulting enolpyruvate then reacts with CO2 to form oxaloacetate and complete the reaction.
登录
查看更多内容
影响因子：
16.8
作者：
Xiang, Song;Tong, Liang
通讯作者：
Tong, Liang
影响因子：
2.9
作者：
Branson, JP;Nezic, M;Attwood, PV
通讯作者：
Attwood, PV
影响因子：
2.9
作者：
CHEUNG, YF;WALSH, C
通讯作者：
WALSH, C
影响因子：
4.1
作者：
CHAPMANSMITH, A;TURNER, DL;WALLACE, JC
通讯作者：
WALLACE, JC
DOI：
10.1042/bj20080709
发表时间：
2008-08-01
期刊：
The Biochemical journal
影响因子：
--
作者：
Jitrapakdee S;St Maurice M;Rayment I;Cleland WW;Wallace JC;Attwood PV
通讯作者：
Attwood PV