Rhodococcus L-phenylalanine dehydrogenase: Kinetics, mechanism, and structural basis for catalytic specifity

Phenylalanine dehydrogenase catalyzes the reversible, pyridine nucleotide-d ependent oxidative deamination of L-phenylalanine to form phenylpyruvate an d ammonia. We have characterized the steady-state kinetic behavior of the e nzyme from Rhodococcus sp. M4 and determined the X-ray crystal structures o f the recombinant enzyme in the complexes, E NADH L-phenylalanine and E NAD (+) L-3-phenyllactate, to 1.25 and 1.4 Angstrom resolution, respectively. I nitial velocity, product inhibition, and dead-end inhibition studies indica te the kinetic mechanism is ordered, with NAD(+) binding prior to phenylala nine and the products' being released in the order of ammonia, phenylpyruva te, and NADH. The enzyme shows no activity with NADPH or other 2'-phosphory lated pyridine nucleotides but has broad activity with NADH analogues. Our initial structural analyses of the E NAD(+) phenylpyruvate and E NAD(+) 3-p henylpropionate complexes established that Lys78 and Asp118 function as the catalytic residues in the active site [Vanhooke et al. (1999) Biochemistry 38, 2326-2339]. We have studied the ionization behavior of these residues in steady-state turnover and use these findings in conjunction with the str uctural data described both here and in our first report to modify our prev iously proposed mechanism for the enzymatic reaction. The structural charac terizations also illuminate the mechanism of the redox specificity that pre cludes alpha-amino acid dehydrogenases from functioning as alpha-hydroxy ac id dehydrogenases.