Alanine dehydrogenase from Enterobacter aerogenes: Purification, characterization, and primary structure

Alanine dehydrogenase [EC 1. 4. 1. 1] was purified to homogeneity from a cr ude extract of Enterobacter aerogenes ICR 0220. The enzyme had a molecular mass of about 245 kDa and consisted of six identical subunits. The enzyme s howed maximal activity at about pH 10.9 for the deamination of L-alanine an d 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 d inucleotide served as coenzymes. Initial-velocity and product inhibition st udies suggested that the deamination of L-alanine proceeded through a seque ntial ordered binary-ternary mechanism. NAD(+) bound first to the enzyme, f ollowed by L-alanine, and the products were released in the order of ammoni a, pyruvate, and NADH. The K-m 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 K -m 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 n ucleotides 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 Enterobac ter enzyme is different from other alanine dehydrogenases.