Large-scale purification and some properties of malate synthase from baker's yeast.
1. Malate synthase from baker's yeast (5 kg) was purified 400--500-fold to homogeneity. About 50--200 mg homogeneous enzyme were obtained within a week in a yield of 30% with reference to the total activity in cell-free crude extracts. The enzyme, pI = 7.5, was pure as judged from ultracentrifugal and gel electrophoretic studies. 2. Sedimentation and diffusion coefficients were determined: S 0 20,w = 8.26 +/- 0.05 S, D 0 20,w = 4.5 +/- 0.1 X 10(-7) cm2 s-1. The molecular weight of the synthase was found to be 175 000 +/- 10 000 and 180 000 +/- 10 000 by sedimentation/diffusion and by high-speed sedimentation equilibrium respectively. It was concluded from these and other results that malate synthase has a molecular mass of 180 000 +/- 10 000. 3. The synthase on sodium dodecylsulfate gel electrophoresis was dissociated to yield a single specimen of Mr about 66 000. The result indicates a composition of the native enzyme from three subunits of identical or nearly identical mass. 4. The binding of acetyl-coenzyme A to the synthase is independent of Mg2+ but that of glyoxylate is strictly dependent on the presence of Mg2+. Kinetic studies indicate that the malate synthase reaction follows a sequential random mechanism. 5. The intermolecular isotopic effect, kH:k2H = 1.4, was determined with acetyl-coenzyme A and [2H3]acetyl-coenzyme A under several different experimental conditions and was shown to reflect different maximal velocities of the two substrates. An enzymic procedure for the preparation of doubly labelled [3H, 14C]acetyl-coenzyme A is also presented.
D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008292:Malate Synthase / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D007652:Oxo-Acid-Lyases / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D046911:Macromolecular Substances; D008292:Malate Synthase; D007652:Oxo-Acid-Lyases; D008274:Magnesium