The International Journal of Biochemistry & Cell Biology 40 (2008) 1743–1752 Available online at www.sciencedirect.com Insights into the mechanism and regulation of pyruvate carboxylase by characterisation of a biotin-deficient mutant of the Bacillus thermodenitrificans enzyme Abdussalam Adina-Zada a , Sarawut Jitrapakdee b , Kathy H. Surinya c , Matthew J. McIldowie a , Matthew J. Piggott a , W. Wallace Cleland d , John C. Wallace c , Paul V. Attwood a,∗ a School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia b Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand c School of Molecular Biosciences, University of Adelaide, Adelaide, SA 5005, Australia d Institute for Enzyme Research and Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53726, USA Received 8 December 2007; accepted 2 January 2008 Available online 6 January 2008 Abstract Pyruvate carboxylase is a biotin-dependent enzyme in which the biotin is carboxylated by a putative carboxyphosphate interme- diate 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 K m s 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 phospho- rylation 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. Keywords: Pyruvate carboxylase; ATP-cleavage; Biotin carboxylation; Acetyl CoA regulation; Stopped-flow ∗ Corresponding author at: Biochemistry and Molecular Biology (M310), School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. Tel.: +61 8 6488 3329; fax: +61 8 6488 1148. E-mail addresses: abdul.adina-zada@uwa.edu.au (A. Adina-Zada), scsji@mucc.mahidol.ac.th (S. Jitrapakdee), kathy.surinya@adelaide.edu.au (K.H. Surinya), mjm@cyllene.uwa.edu.au (M.J. McIldowie), piggott@cyllene.uwa.edu.au (M.J. Piggott), cleland@enzyme.wisc.edu (W.W. Cleland), john.wallace@adelaide.edu.au (J.C. Wallace), pattwood@cyllene.uwa.edu.au (P.V. Attwood). 1357-2725/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2008.01.001