Utilization of oxalacetate by Acinetobacter calcoaceticus: evidence for coupling between malic enzyme and malic dehydrogenase.

Growth of Acinetobacter calcoaceticus strain BD413 in malate-mineral medium resulted in the excretion of large quantities of oxalacetate. Malate was virtually depleted by the time the cell density reached 60% of its final value; most of the remaining growth took place at the expense of oxalacetate. Experiments in which oxalacetate was used as the initial substrate showed that pyruvate was not utilized until most of the oxalacetate disappeared. The generation time for growth on malate or oxalacetate was approximately 40 min; the generation time for growth on pyruvate was 62 min, which implies that pyruvate transport may be rate limiting. Oxalacetate and pyruvate, however, supported approximately the same growth yield. These observations suggested that the first step in the utilization of oxalacetate as an energy source consisted of an enzymatic decarboxylation of the keto acid to pyruvate and CO(2). Three enzyme reactions that carry out this decarboxylation have been detected in extracts of A. calcoaceticus. The first, which functioned maximally at pH 4.8, was attributable to the oxalacetate decarboxylase activity of oxidized diphosphopyridine nucleotide-malic enzyme. The second and third, which functioned in the neutral pH range, resulted from coupling of oxidized diphosphopyridine nucleotide-malic enzyme to reduced diphosphopyridine nucleotide-dependent malic dehydrogenase, and oxidized triphosphopyridine nucleotide-malic enzyme to a reduced triphosphopyridine nucleotide-dependent malic dehydrogenase. The efficiency of these coupled reactions was high enough so that the overall reaction could be physiologically significant.

Reduced diphosphopyridine nucleotide peroxidase. Intermediates formed on reduction of the enzyme with dithionite or reduced diphosphopyridine nucleotide.

DPNH peroxidase is a flavin adenine dinucleotide-containing flavoprotein. Anaerobic titration of enzyme with dithionite has shown that the active site of the enzyme contains 2 mol of flavin and in addition 1 mol of a non-flavin electron acceptor that is tentatively identified as a disulfide group. Thus complete reduction of the enzyme requires 3 mol of dithionite per mole of active site. The first mole of dithionite reduces the non-flavin acceptor; complex formation between the reduced acceptor and one of the bound flavin molecules causes the formation of a long wavelength absorption band between 500 and 670 nm. The second mole of dithionite reduces the flavin that interacts with the reduced non-flavin group, and the long wavelength band disappears. The third mole of dithionite reduces the second mole of flavin. All groups are reoxidized in the presence of air. DPNH reacts with only two of the enzyme-bound electron acceptors. The first mole of DPNH reduces the non-flavin group to form an intermediate (I) that is almost identical with that formed by dithionite. The second mole of DPNH complexes with the second flavin of Intermediate I to form Intermediate II. This reaction causes a further absorbance increase in the long wavelength region; the tail of the absorption band now extends to 960 nm. The titration data (potassium phosphate, 0.05 M, pH 7.0) can be fitted with dissociation constants of 1 times 10-7 M for the formation of I, and 3 times 10-6 M for the conversion of I to II. In air, species II is oxidized to I; I is stable in air, but is oxidized stoichiometrically to oxidized enzyme by H2O2. Present evidence suggests that bound DPN-plus is responsible for the air stability of species I. Intermediate I, but not oxidized enzyme, reacts slowly with phenylmercuric acetate. This reaction causes loss of the air-stable intermediate and parallel loss in enzyme activity. The inactive enzyme cannot be reduced by DPNH to Species I; DPNH can, however, still react with the second flavin to form the autoxidizable complex. With other methods of enzyme inactivation there is also a direct correlation between residual enzyme activity and the ability of enzyme to form the air-stable intermediate. It is concluded that the air-stable intermediate is an important catalytic species.