Purification, characterisation and mutagenesis of highly expressed recombinant yeast pyruvate kinase

Abstract

Recombinant yeast pyruvate kinase has been purified from a strain of Saccharomyces cerevisiae expressing the enzyme to very high levels. Expression was from a multicopy plasmid under the control of the yeast phosphoglycerate kinase promoter. The gene was expressed in the absence of the genomically encoded pyruvate kinase, using a strain of yeast in which the pyruvate kinase gene has been disrupted by the insertion of the yeast Ura3 gene. The purification procedure minimised proteolytic artefacts and enabled the covenient purification of 15 -20 mg enzyme from 1 1 culture. The purified enzyme was characterised by a high specific activity and by a lack of proteolytic degradation. Two active-site mutants of yeast pyruvate kinase have been produced, expressed and characterised in this system and preliminary results are described.Pyruvate kinase (ATP : pyruvate 2-0-phosphotransferase) is an enzyme of glycolysis that plays a major role in regulating the flux from fructose 1,6-bisphosphate (Fru(l,6)P2) to pyruvate. The enzyme is a homotetramer, with a subunit M , of 55 000 -60000, and catalyses the essentially irreversible reaction,Pyruvate kinase has been extensively studied from a wide range of organisms and much is known about its physical and catalytic properties. It exists as four isoenzymes in mammals designated M1, M2, L and R. The M1 isoenzyme is essentially non-regulated, whereas the remaining isoenzymes (as well as the enzymes from lower organisms) exhibit a sigmoidal kinetic profile with respect to phosphoenolpyruvate concentration, and are allosterically regulated by a large number of nonsubstrate molecules. All forms of the enzyme require both monovalent and bivalent cations for activity. The former is normally potassium whereas a number of bivalent cations will suffice. Two bivalent cations are required/active site, one primarily associated with the nucleotide and the other more closely enzyme associated (see Muirhead, 1987, for a review).The three-dimensional structure of cat muscle M1 isoenzyme has been solved to a resolution of 0.26 nm (Muirhead et al., 1986). Each subunit of cat muscle M1 pyruvate kinase consists of four domains, N, A, B and C. Domain A is an eight-stranded a/B barrel, typical of a number of enzymes (Farber and Petsko, 1990). The active site lies between do-1 Kt, Mg'+

The cooperative binding of fructose-1,6-bisphosphate to yeast pyruvate kinase.

“…Preparation of yeast pyruvate kinase Pyruvate kinase was prepared from a transformed strain of S.cerevisiae overexpressing yeast pyruvate kinase to very high levels according to Murcott et al (1991).…”

“…Fluorometric titration Fluorescence titrations were performed according to Murcott et al (1991) with the following modifications. The buffer used was Triethanolamine-HCL pH 6.2 and the titrations were perforned in a Perkin Elmer LS-5B fluorimeter and with the aid of a Harvard Instruments Pump-22 microprocessor controlled titrator.…”

Population structure and gene evolution inSaccharomyces cerevisiae

“…The four genes examined are located on four different chromosomes and perform varied functions. The gene CDC19 is a housekeeping gene coding for pyruvate kinase, a metabolic enzyme of key importance to the yeast cell cycle (Murcott, 1991). The low density of single-nucleotide polymorphisms in the coding region indicates a high level of conservation in this gene ( Table 6).…”

Mechanism of proanthocyanidins-induced alcoholic fermentation enhancement inSaccharomyces cerevisiae

“…The mixtures were centrifuged at 4 °C at 12,000 rpm for 10 min to obtain the crude enzymes. The activities of HK (a, b), PKF (c, d) and PK (e, f) were measured according to the methods described by Martinez-Barajas and Randall [19], Bär et al [3] and Murcott et al [23] with slight modifications, respectively. Data were shown as the mean ± SD of three independent experiments Facilitated diffusion is the main mechanism of the sugar uptakes in yeast cells under high sugar concentration [33].…”

“…The mixtures were centrifuged at 4 °C at 12,000 rpm for 10 min to obtain the crude enzymes. The activities of the hexokinase, phosphofructokinase and pyruvate kinase were measured as described by Bär et al [3], Murcott et al [23] and Martinez-Barajas, Randall [19] with some slight modifications, respectively, and expressed as μmol products min −1 mg −1 protein.…”