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Pyruvate kinase (Pyk1) levels influence both the rate and direction of carbon flux in yeast under fermentative conditions

Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK¹
Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504 UR-INRA 792, Département de Génie Biochimique et Alimentaire, Institut National des Sciences Appliquées, 31077 Toulouse Cedex 04, France²
Department of Chemistry, Cardiff University, PO Box 912, Cardiff CF10 3TB, UK³
Cardiff School of Biosciences, Cardiff University, PO Box 915, Cardiff CF10 3TL, UK⁴

Author for correspondence: Alistair J. P. Brown. Tel: +44 1224 273183. Fax: +44 1224 273144. e-mail: al.brown{at}abdn.ac.uk

Yeast phosphofructo-1-kinase (Pf1k) and pyruvate kinase (Pyk1) are allosterically regulated enzymes that catalyse essentially irreversible reactions in glycolysis. Both the synthesis and activity of these enzymes are tightly regulated. To separate experimentally the control of Pf1k and Pyk1 synthesis from their allosteric regulation, a congenic set of PFK1, PFK2 and PYK1 mutants was constructed in which these wild-type coding regions were driven by alternative promoters. Mutants carrying PGK1 promoter fusions displayed normal rates of growth, glucose consumption and ethanol production, indicating that the relatively tight regulation of Pyk1 and Pf1k synthesis is not essential for glycolytic control under fermentative growth conditions. Mutants carrying fusions to an enhancer-less version of the PGK1 promoter (PGK1₇₆₇) expressed Pyk1 and Pf1k at about 2·5-fold lower levels than normal. Physiological and metabolic analysis of the PFK1 PFK2 double mutant indicated that decreased Pf1k had no significant effect on growth, apparently due to compensatory increases in its positive effector, fructose 2,6-bisphosphate. In contrast, growth rate and glycolytic flux were reduced in the PGK1₇₆₇–PYK1 mutant, which had decreased Pyk1 levels. Unexpectedly, the reduced Pyk1 levels caused the flow of carbon to the TCA cycle to increase, even under fermentative growth conditions. Therefore, Pyk1 exerts a significant level of control over both the rate and direction of carbon flux in yeast.

Keywords: Glycolysis, metabolic flux, pyruvate kinase, phosphofructokinase, yeast physiology

Abbreviations: MCA, metabolic control analysis; Pf1k, 6-phosphofructo-1-kinase; Pyk1, pyruvate kinase

^a Present address: MRC Radiation and Genome Stability Unit, Harwell, Didcot, Oxfordshire OX11 ORD, UK.

^b Present address: Quest International, Menstrie, Clackmannanshire SK11 7ES, UK.

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