Purification and Characterization of an Enantioselective Amidase from Pseudomonas chlororaphis B23.

An amidase produced by Pseudomonas chlororaphis B23 was purified and characterized. The purification procedure used included ammonium sulfate precipitation and hydrophobic, anion-exchange, gel filtration, and ceramic hydroxyapatite chromatography steps. This amidase has a native molecular mass of about 105 kDa and is a homodimer whose subunits have a molecular mass of 54 kDa. The enzyme exhibited maximal activity at 50(deg)C and at pH values ranging from 7.0 to 8.6. We found no evidence that metal ions were required, and the enzyme was inhibited by several thiol reagents. This amidase exhibited activity against a broad range of aliphatic and aromatic amides and exhibited enantioselectivity for several aromatic amides, including 2-phenylpropionamide (enantiomeric excess [ee] = 100%), phenylalaninamide (ee = 55%), and 2-(4-chlorophenyl)-3-methylbutyramide (ee = 96%), but not 2-(6-methoxy-2-naphthyl)propionamide (the amide form of naproxen) (ee = 0%). The characteristics of the P. chlororaphis B23 amidase are the same as the characteristics of enantioselective amidases described by Mayaux et al. (J. F. Mayaux, E. Cerbelaud, F. Soubrier, D. Faucher, and D. Petre, J. Bacteriol. 172:6764-6773, 1990; J. F. Mayaux, E. Cerbelaud, F. Soubrier, P. Yeh, F. Blanche, and D. Petre, J. Bacteriol. 173:6694-6704, 1991) and Kobayashi et al. (M. Kobayashi, H. Komeda, T. Nagasawa, M. Nishiyama, S. Horinouchi, T. Beppu, H. Yamada, and S. Shimizu, Eur. J. Biochem. 217:327-336, 1993).

Investigations of the partial reactions catalyzed by pyruvate phosphate dikinase.

The kinetic mechanism of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus was investigated with several different kinetic diagnostics. Initial velocity patterns were intersecting for AMP/PPi and ATP/Pi substrate pairs and parallel for all other substrate pairs. PPDK was shown to catalyze [14C]pyruvate in equilibrium phosphoenolpyruvate (PEP) exchange in the absence of cosubstrates, [14C]AMP in equilibrium ATP exchange in the presence of Pi/PPi but not in their absence, and [32P]Pi in equilibrium PPi exchange in the presence of ATP/AMP but not in their absence. The enzyme was also shown, by using [alpha beta-18O, beta, beta-18O2]ATP and [beta gamma-18O, gamma, gamma, gamma-18O3]ATP and 31P NMR techniques, to catalyze exchange in ATP between the alpha beta-bridge oxygen and the alpha-P nonbridge oxygen and also between the beta gamma-bridge oxygen and the beta-P nonbridge oxygen. The exchanges were catalyzed by PPDK in the presence of Pi but not in its absence. These results were interpreted to support a bi(ATP,Pi) bi(AMP,PPi) uni(pyruvate) uni(PEP) mechanism. AMP and Pi binding order was examined by carrying out dead-end inhibition studies. The dead-end inhibitor adenosine 5'-monophosphorothioate (AMPS) was found to be competitive vs AMP, noncompetitive vs PPi, and uncompetitive vs PEP. The dead-end inhibitor imidodiphosphate (PNP) was found to be competitive vs PPi, uncompetitive vs AMP, and uncompetitive vs PEP. These results showed that AMP binds before PPi. The ATP and Pi binding order was studied by carrying out inhibition, positional isotope exchange, and alternate substrate studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition and inactivation of pyruvate phosphate dikinase with Cr(III) complexes of adenosine 5'-triphosphate and inorganic pyrophosphate.

The exchange inert complexes beta,gamma-bidentate Cr(H2O)4ATP and P1,P2-bidentate Cr(H2O)4PP were found to bind to the Bacteriodes symbiosus pyruvate phosphate dikinase ATP and PP binding sites, respectively. The inactivation of the enzyme that was observed with these complexes was shown to involve covalent attachment of the entire complex to the enzyme via insertion of enzyme amino acid side chains into the coordination sphere of the Cr(III). Incubation of Cr(H2O)4ATP with other proteins also resulted in covalent attachment.