Biosynthesis, isolation and properties of NAD-dependent formate dehydrogenase from the yeast Candida methylica.

NAD-dependent formate dehydrogenase (EC 1.2.1.2), was isolated from the methanol-utilizing yeast Candida methylica. Two purification techniques for the enzyme from the crude yeast extract have been developed: a two-step procedure, involving a sequential application of DEAE-cellulose ion-exchange chromatography and Sephadex G-200 gel filtration, and a single-step procedure, preparative isoelectric focusing in a granulated gel layer. The enzyme proved to be electrophoretically homogeneous. It consisted of two identical subunits with a relative molecular mass of 46 000, each containing one -SH group related to manifestation of the catalytic activity. The Michaelis constant was 1 X 10(-4) M for NAD and 1.3 X 10(-2) M for formate. Formate dehydrogenase was inhibited with p-chlormercuribenzoate, iodoacetamide, dithionitrobenzoate, cyanide and azide.

[Kinetics of NAD-dependent formate dehydrogenase from the methanol-utilizing yeast Candida methylica]. / Kineticheskii mekhanizm deistviia NAD-zavisimoi formiatdegidrogenazy metilotrofnykh drozhzhei Candida methylica.

A kinetic analysis of the mechanism of action of NAD-dependent formate dehydrogenase (EC 1.2.1.2) from the methanol-utilizing yeast Candida methylica has been carried out. The dependence of the initial reaction rate on substrate concentrations and the inhibition by the reaction products and substrate analogs were investigated. The data obtained suggest that the kinetics of the formate dehydrogenase action are consistent with the formation of a ternary enzyme--substrate complex. NAD is the first substrate and NADH is the last product of the reaction, respectively.

[Isolation and properties of NAD-dependent formate dehydrogenase from the yeast Candida methylica]. / Vydelenie i svoistva NAD-zavisimoi formiatdegidrogenazy iz drozhzhei Candida methylica.

NAD-dependent formate dehydrogenase was purified from a cell-free extract of methanol-consuming yeast Candida methylica, using chromatography on DEAE-cellulose and gel-filtration on Sephadex G-200. The enzyme is electrophoretically homogeneous, consists of two identical subunits with molecular weight of 46,000 and is active within the pH range of 6-9; the Km values for NAD and formate are 1 . 10(-4) and 1,3 . 10(-2) M, respectively. Formate dehydrogenase is inhibited by p-chloromercurybenzoate, iodoacetate, dithionitrobenzoate and azide.

NAD-dependent formate dehydrogenase from methylotrophic bacterium, strain 1. Purification and characterization.

1. NAD-dependent formate dehydrogenase was isolated from gram-negative methylotrophic bacteria, strain 1, grown on methanol. The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography and preparative isotachophoresis or gel filtration; it resulted in a yield of 40%. 2. The final enzyme preparations were homogeneous as judged by sedimentation in an ultracentrifuge. Formate dehydrogenase purified in the presence of EDTA reveals two bands on electrophoresis in polyacrylamide gel both after protein and activity staining. Two components are transformed into a single one after prolonged storage in the presence of 2-mercaptoethanol. 3. Formate dehydrogenase is a dimer composed of identical or very similar subunits. The molecular weight of the enzyme is about 80 000. 4. Amino acid composition and some other physico-chemical properties of the enzyme were studied. 5. Formate dehydrogenase is specific for formate and NAD as electron acceptor. The Michaelis constant was 0.11 mM for NAD and 15 mM for formate (pH 7.0, 37 degrees C). 6. Formate dehydrogenase was rapidly inactivated in the absence of -SH compounds. The enzyme retained full activity upon storage at ambient temperature in solution for half a year in the presence of 2-mercaptoethanol or EDTA.

[Nad-dependent formate dehydrogenase from methylotrophic bacteria. Characteristics and stability of soluble and immobilized enzyme]. / NAD-zavisimaia formiatdegidrogenaza iz metilotrofnykh bakterii. Izuchenie svoistv i stabil'nosti rastvorimogo i immobilizovannogo fermenta.

pH-dependency is studied of kinetic parameters of the reaction catalyzed by NAD-dependent formate dehydrogenase from methylotrophic Bacterium spl strain. Values of Km for NAD and formate, and also of maximum reaction rate are found not to change within the pH range from 6 to 9. Role of SH-groups in the development of the enzyme catalytic activity and the effect of different factors on stability of soluble and immobilized enzyme forms are investigated. Molecular weight of the enzyme (70000), extinction coefficient and catalytical constant (6 s-1) are determined.

[NAD-dependent formate dehydrogenase from methylotrophic bacteria. Isolation and properties]. / NAD-zavisimaia formiatdegidrogenaza iz metilotrofnykh bakterii. Ochistka i osnovnye svoistva.

NAD-Dependent formate dehydrogenase (FDH) has been isolated from methylotrophyc strain Bacterium sp 1 by (NH4)2SO4 fractionation of cell extract, ion-exchange chromatography and preparative isotachophoresis. Preparation of FDH is homogeneous in analytical polyacrylamide gel electrophoresis and under ultracentrifugation. Sedimentation coefficient of FDH is 4.9S. Mikhaelis constants are 1.1-10(-4) M for NAD and 1.5-10(-2) M for formate. In the absence of sulfhydril compounds FDH is unstable, but it is stable in the presence of mercaptoethanol or ditiotreitol.