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PEDS Advance Access originally published online on March 23, 2005
Protein Engineering Design and Selection 2005 18(2):71-78; doi:10.1093/protein/gzi011
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions{at}oupjournals.org

Construction of new forms of pyruvate carboxylase to assess the allosteric regulation by acetyl-CoA

M.Nurul Islam, Shinji Sueda1 and Hiroki Kondo

Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Kawazu 680-4, Iizuka 820-8502, Japan

1 To whom correspondence should be addressed. E-mail: sueda{at}bio.kyutech.ac.jp

The single polypeptide chain of Bacillus thermodenitrificans pyruvate carboxylase (PC) is composed of the biotin carboxylase (BC), carboxyl transferase (CT) and biotin carboxyl carrier protein (BCCP) domains from the amino terminus. This polypeptide chain was divided into two between the CT and BCCP domains. The resulting proteins, PC-(BC + CT) and PC-(BCCP), were expressed in Escherichia coli separately, purified to homogeneity and characterized. PC-(BC + CT) was 4% as active as native PC in the carboxylation of pyruvate with PC-(BCCP) as substrate with a Km of 39 µM. Moreover, acetyl-CoA stimulated the carboxylation of PC-(BCCP) about 3-fold, whereas it was without effect in the corresponding reaction with free biotin. In addition to these engineered proteins, another form of enzyme was also constructed in which the BC domain of B.thermodenitrificans PC was replaced with the BC subunit of Aquifex aeolicus PC, whose activity is independent of acetyl-CoA. The resulting chimera was about 7% as active as native PC, but its activity was independent of acetyl-CoA. On the basis of these observations, the mechanism by which acetyl-CoA regulates the reaction of PC is discussed.

Received November 5, 2004; revised January 31, 2005; accepted February 14, 2005.

Edited by Anthony Wilkinson


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