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'+