Mechanistic and genetic analysis of phenylalanine dehydrogenase

Abstract

Phenylalanine dehydrogenase (EC 1.4.1.20) catalyzes the reversible deamination of L-phenylalanine with the concomitant reduction of NAD. This enzyme is the most recent addition to the family of amino acid dehydrogenases.;Using a variety of chromatographic techniques, the protein was purified to apparent homogeneity from Rhodococcus sp. M4, a bacterium which expresses the enzyme at high levels when induced with L-phenylalanine. The pdh gene was cloned from a cosmid library created from genomic DNA, and overexpressed in E. coli. The gene sequence was determined and extensive sequence comparisons were made to other amino acid dehydrogenases. Strong similarities are seen in the dinucleotide binding domains. The catalytic domains show fewer similarities but include conserved residues which are important in amino acid binding and discrimination.;Initial velocity and inhibition experiments were used in order to determine the steady-state kinetic mechanism. The enzyme exhibited a sequential order of substrate addition and product release, with NAD binding first followed by L-phenylalanine, and with ammonia, phenylpyruvate, and NADH released in that order. The stereochemistry of hydride transfer was determined to be pro-S by mass spectrometry.;The pK values of enzymic groups involved in substrate binding and in catalysis were determined by measuring the pH dependence of the kinetic constants, V/K and V, respectively. In addition, the ionization state of the substrates required for effective binding are determined using this technique. Primary deuterium isotope effects were measured in the forward and reverse directions. The isotope effects on {dollar}\sp{lcub}\rm D{rcub}{dollar}V/K{dollar}\sb{lcub}\rm phe{rcub}{dollar}, {dollar}\sp{lcub}\rm D{rcub}{dollar}V/K{dollar}\sb{lcub}\rm amm{rcub}{dollar}, and {dollar}\sp{lcub}\rm D{rcub}{dollar}V are small (ca. 1.25). These isotope effects are somewhat larger when alternate nucleotide substrates were used. Solvent deuterium isotope effects were also small in both the forward and reverse reactions (1.34 and 1.78, respectively). Natural abundance {dollar}\sp{lcub}15{rcub}{dollar}N isotope effects were measured by isotope ratio mass spectrometry over a range of pH values. At low pH values, the {dollar}\sp{lcub}15{rcub}{dollar}N isotope effect is inverse (0.980), but approaches unity as the pH values are increased.;From these data, we propose a model for the catalytic mechanism, including groups involved in catalysis and the relative rates of catalytic steps.