Publication: Conserved Glu40 and Glu433 of the biotin carboxylase domain of yeast pyruvate carboxylase I isoenzyme are essential for the association of tetramers
Issued Date
2007-09-11
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ISSN
13572725
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2-s2.0-34548457403
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Mahidol University
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SCOPUS
Bibliographic Citation
International Journal of Biochemistry and Cell Biology. Vol.39, No.11 (2007), 2120-2134
Suggested Citation
Sarawut Jitrapakdee, Katharina H. Surinya, Abdussalam Adina-Zada, Steven W. Polyak, Cvetan Stojkoski, Roger Smyth, Grant W. Booker, W. Wallace Cleland, Paul V. Attwood, John C. Wallace Conserved Glu40 and Glu433 of the biotin carboxylase domain of yeast pyruvate carboxylase I isoenzyme are essential for the association of tetramers. International Journal of Biochemistry and Cell Biology. Vol.39, No.11 (2007), 2120-2134. doi:10.1016/j.biocel.2007.06.006 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/24119
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Title
Conserved Glu40 and Glu433 of the biotin carboxylase domain of yeast pyruvate carboxylase I isoenzyme are essential for the association of tetramers
Abstract
The native form of pyruvate carboxylase is an α4tetramer but the tetramerisation domain of each subunit is currently unknown. To identify this domain we co-expressed yeast pyruvate carboxylase 1 isozyme (Pyc1) with an N-terminal myc tag, together with constructs encoding either the biotin carboxylase (BC) domain or the transcarboxylase-biotin carboxyl carrier domain (TC-BCC), each with an N-terminal 9-histidine tag. From tag-affinity chromatography experiments, the subunit contacts within the tetramer were identified to be primarily located in the 55 kDa BC domain. From modelling studies based on known structures of biotin carboxylase domains and subunits we have predicted that Arg36 and Glu433 and Glu40 and Lys426, respectively, are involved pairwise in subunit interactions and are located on opposing subunits in the putative subunit interface of Pyc1. Co-expression of mutant forms with wild type Pyc1 showed that the R36E mutation had no effect on the interaction of these subunits with those of wild type Pyc1, while the E40R, E433R and R36E:E433R mutations caused severe loss of interaction with wild type Pyc1. Ultracentrifugal analysis of these mutants when expressed and purified separately indicated that the predominant form of E40R, E433R and R36R:E433R mutants is the monomer, and that their specific activities are less than 2% of the wild type. Studies on the association state and specific activity of the R36E mutant at different concentrations showed it to be much more susceptible to tetramer dissociation and inactivation than the wild type. Our results suggest that Glu40 and Glu433 play essential roles in subunit interactions.