Alanine dehydrogenase from Enterobacter aerogenes: purification, characterization, and primary structure.
Alanine dehydrogenase [EC 1. 4. 1. 1] was purified to homogeneity from a crude extract of Enterobacter aerogenes ICR 0220. The enzyme had a molecular mass of about 245 kDa and consisted of six identical subunits. The enzyme showed maximal activity at about pH 10.9 for the deamination of L-alanine and at about pH 8.7 for the amination of pyruvate. The enzyme required NAD+ as a coenzyme. Analogs of NAD+, deamino-NAD+ and nicotinamide guanine dinucleotide served as coenzymes. Initial-velocity and product inhibition studies suggested that the deamination of L-alanine proceeded through a sequential ordered binary-ternary mechanism. NAD+ bound first to the enzyme, followed by L-alanine, and the products were released in the order of ammonia, pyruvate, and NADH. The Km were 0.47 mM for L-alanine, 0.16 mM for NAD+, 0.22 mM for pyruvate, 0.067 mM for NADH, and 66.7 mM for ammonia. The Km for L-alanine was the smallest in the alanine dehydrogenases studied so far. The enzyme gene was cloned into Escherichia coli JM109 cells and the nucleotides were sequenced. The deduced amino acid sequence was very similar to that of the alanine dehydrogenase from Bacillus subtilis. However, the Enterobacter enzyme has no cysteine residue. In this respect, the Enterobacter enzyme is different from other alanine dehydrogenases.
Chowdhury
E K
EK
Department of Bioresources Science, Kochi University, Japan.
Saitoh
T
T
Nagata
S
S
Ashiuchi
M
M
Misono
H
H
D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003067:Coenzymes / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004754:Enterobacter / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry; D013379:Substrate Specificity