Abstract
NAD+-dependent phenylalanine dehydrogenases were purified 1,500- and 1,600-fold, and crystallized from Sporosarcina ureae SCRC-R04 and Bacillus sphaericus SCRC-R79a, respectively. The purified enzymes were homogeneous as judged by disc gel electrophoresis. The enzyme from S. ureae has a molecular weight of 305,000, while that of B. sphaericus has a molecular weight of 340,000. Each is probably composed of eight subunits identical in molecular weight. The S. ureae enzyme showed a high substrate specificity in the oxidative deamination reaction acting on L-phenylalanine, while that of B. sphaericus acted on L-phenylalanine and L-tyrosine. The enzymes had lower substrate specificities in the reductive amination reaction acting on alpha-keto acids. The Sporosarcina enzyme acted on phenylpyruvate, alpha-ketocaproate, alpha-keto-gamma-methylthiobutyrate and rho-hydroxyphenylpyruvate. The Bacillus enzyme acted on rho-hydroxyphenylpyruvate, phenylpyruvate, and alpha-keto-gamma-methylthiobutyrate. The enzyme from B. sphaericus catalyzes The enzyme from B. sphaericus catalyzes the transfer of pro-S (B) hydrogen from NADH.
Highlights
Immunotitrations were ammonium sulfate carried out using as an antigen the purified enzyme or crude prepa- DEAE-Toyopearl
1,910 3,890 2.04 rations obtained by the ammonium sulfate fractionation
Summary of purification of phenylalanine dehydrogenase from B. sphaericus SCRC-R79a was prepared by reductive amination of a-ketoisocaproate with a The concentration of the purified enzyme after the DEAE-Toyoregeneration of dideuterated NADH using alcohol dehydrogenase and pearl step was determined from the absorbance at 280 nm using the aldehyde dehydrogenase with [U-'H]ethanol as asubstrate
Summary
From the Sagami Chemical ResearchCenter, Nishi-Ohnumn 4-4-1, Sagamihura, Kanagawa 229, Japan. The S. ureae enzyme showed a high substrate specificity in the oxidative deamination reaction acting on Lphenylalanine, while that of B. sphaericus acted on L-. Benner andcoworkers [14, 15] have recently demonstrated that the stereochemistry indehydrogenases acting onalcohols follows a rule; the stabilityof the ketone in the oxidation-reduction reactions determinesthe stereochemicapl reference inthe hydride transfer. These situationshave prompted us to study phenylalanine dehydrogenase. This paper describes the purification to homogeneity and physical, catalytic, and immunological properties of the enzyme from S. ureae and B. sphaericus, in comparison with other aminoacid dehydrogenases
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