Studies on the allosteric control of yeast pyruvate kinase

• Main Author: McNally, Teresa
• Published: University of Edinburgh 1991
• Subjects: 572.7
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.657095

Pyruvate kinase [EC.2.7.1.40.] is ubiquitous and catalyses the second of the ATP forming reactions of glycolysis, namely the interconversion of phosphoenolpyruvate to pyruvate with concomitant phosphotransfer to ADP to form ATP. The enzyme requires one monovalent and two divalent cations for full activity, one of which is enzyme bound and the others being abstracted to the nucleotide. The enzyme is considered to be the control point for the lower part of the glycolytic pathway. Pyruvate kinases can be allosterically regulated by a number of effectors which bind to the enzyme at sites distinct from the substrate binding site and modify the enzyme activity. It was the purpose of this project to attempt to determine the nature of this form of regulation using site directed mutagenesis to alter residues to be involved in mediating the allosteric effect. In this project the pyruvate kinase from the yeast <i>Saccharomyces cerevisiae</i> is used as the model for an allosterically regulated pyruvate kinase. This yeast has only one gene encoding a pyruvate kinase (<i>pyk1</i>) which is subject to allosteric control. This gene had been cloned and sequenced prior to the outset of this project, however the sequence was seen to contain a number of errors which led me to repeat this process. The approach adopted in this project was to overexpress pyruvate kinase on a multicopy yeast shuttle vector, this facilitated its subsequent purification. It is hoped that this approach will facilitate the production of very large amounts of protein which can be used for x-ray crystallography and other physico-chemical techniques which require large amounts of protein.