Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli
The effects of mutations in the active site of the carboxyl transferase domain of Rhizobium 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 Mg...
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th-mahidol.272222018-09-13T13:24:35Z Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli Tonya N. Zeczycki Martin St. Maurice Sarawut Jitrapakdee John C. Wallace Paul V. Attwood W. Wallace Cleland University of Wisconsin Madison, Institute for Enzyme Research Marquette University Mahidol University University of Adelaide University of Western Australia Biochemistry, Genetics and Molecular Biology The effects of mutations in the active site of the carboxyl transferase domain of Rhizobium 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 7- and 3.5-fold increases in activity, as compared to that of 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 removal of a proton 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. © 2009 American Chemical Society. 2018-09-13T06:24:35Z 2018-09-13T06:24:35Z 2009-05-26 Article Biochemistry. Vol.48, No.20 (2009), 4305-4313 10.1021/bi9003759 00062960 2-s2.0-66149189429 https://repository.li.mahidol.ac.th/handle/123456789/27222 Mahidol University SCOPUS https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=66149189429&origin=inward |
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Biochemistry, Genetics and Molecular Biology Tonya N. Zeczycki Martin St. Maurice Sarawut Jitrapakdee John C. Wallace Paul V. Attwood W. Wallace Cleland Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli |
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The effects of mutations in the active site of the carboxyl transferase domain of Rhizobium 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 7- and 3.5-fold increases in activity, as compared to that of 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 removal of a proton 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. © 2009 American Chemical Society. |
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University of Wisconsin Madison, Institute for Enzyme Research |
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University of Wisconsin Madison, Institute for Enzyme Research Tonya N. Zeczycki Martin St. Maurice Sarawut Jitrapakdee John C. Wallace Paul V. Attwood W. Wallace Cleland |
format |
Article |
author |
Tonya N. Zeczycki Martin St. Maurice Sarawut Jitrapakdee John C. Wallace Paul V. Attwood W. Wallace Cleland |
author_sort |
Tonya N. Zeczycki |
title |
Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli |
title_short |
Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli |
title_full |
Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli |
title_fullStr |
Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli |
title_full_unstemmed |
Insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from Rhizobium etli |
title_sort |
insight into the carboxyl transferase domain mechanism of pyruvate carboxylase from rhizobium etli |
publishDate |
2018 |
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https://repository.li.mahidol.ac.th/handle/123456789/27222 |
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1763496306362286080 |