Rhodococcus L-phenylalanine dehydrogenase: kinetics, mechanism, and structural basis for catalytic specificity.

Phenylalanine dehydrogenase catalyzes the reversible, pyridine nucleotide-dependent oxidative deamination of l-phenylalanine to form phenylpyruvate and ammonia. We have characterized the steady-state kinetic behavior of the enzyme from Rhodococcus sp. M4 and determined the X-ray crystal structures of the recombinant enzyme in the complexes, E·NADH·l-phenylalanine and E·NAD+·l-3-phenyllactate, to 1.25 and 1.4 A resolution, respectively. Initial velocity, product inhibition, and dead-end inhibition studies indicate the kinetic mechanism is ordered, with NAD+ binding prior to phenylalanine and the products' being released in the order of ammonia, phenylpyruvate, and NADH. The enzyme shows no activity with NADPH or other 2‘-phosphorylated pyridine nucleotides but has broad activity with NADH analogues. Our initial structural analyses of the E·NAD+·phenylpyruvate and E·NAD+·3-phenylpropionate complexes established that Lys78 and Asp118 function as the catalytic residues in the active site [Vanhooke et al. (199...