Simple jQuery Dropdowns
Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/18988
Title: Insights into the mechanism and regulation of pyruvate carboxylase by characterisation of a biotin-deficient mutant of the Bacillus thermodenitrificans enzyme
Authors: Abdussalam Adina-Zada
Sarawut Jitrapakdee
Kathy H. Surinya
Matthew J. McIldowie
Matthew J. Piggott
W. Wallace Cleland
John C. Wallace
Paul V. Attwood
University of Western Australia
Mahidol University
University of Adelaide
University of Wisconsin Madison, Institute for Enzyme Research
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 25-Jan-2008
Citation: International Journal of Biochemistry and Cell Biology. Vol.40, No.9 (2008), 1743-1752
Abstract: Pyruvate carboxylase is a biotin-dependent enzyme in which the biotin is carboxylated by a putative carboxyphosphate intermediate that is formed in a reaction between ATP and bicarbonate. The resultant carboxybiotin then transfers its carboxyl group to pyruvate to form oxaloacetate. In the Bacillus thermodenitrificans enzyme the biotin is covalently attached to K1112. A mutant form of the enzyme (K1112A) has been prepared which is not biotinylated. This mutant did not catalyse the complete reaction, but did catalyse ATP-cleavage and the carboxylation of free biotin. Oxaloacetate decarboxylation was not catalysed, even in the presence of free biotin, suggesting that only the biotin carboxylation domain of the enzyme is accessible to free biotin. This mutant allowed the study of ATP-cleavage both coupled and not coupled to biotin carboxylation. Kinetic analyses of these reactions indicate that the major effect of the enzyme activator, acetyl CoA, is to promote the carboxylation of biotin. Acetyl CoA reduces the Kms for both MgATP and biotin. In addition, pH profiles of the ATP-cleavage reaction in the presence and absence of free biotin revealed the involvement of several ionisable residues in both ATP-cleavage and biotin carboxylation. K1112A also catalyses the phosphorylation of ADP from carbamoyl phosphate. Stopped-flow studies using the fluorescent ATP analogue, formycin A-5′-triphosphate, in which nucleotide binding to the holoenzyme was compared to K1112A indicated that the presence of biotin enhanced binding. Attempts to trap the putative carboxyphosphate intermediate in K1112A using diazomethane were unsuccessful. © 2008 Elsevier Ltd. All rights reserved.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=49949151841&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/18988
ISSN: 13572725
Appears in Collections:Scopus 2006-2010

Files in This Item:
There are no files associated with this item.


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.