Reaction Mechanism of d-Galactose Dehydrogenases from Pseudomonas saccharophila and Pseudomonas fluorescens. Formation and Rearrangement of Aldono-1,5-lactones

Abstract

1. b-D-Galactopyranose has been shown by specificity studies and by gas-liquid chromatographic investigations to be the substrate of the D-galactose dehydrogenases from Pseudomonas sacchurophilu and from Pseudomonas fluorescens.2. cc-~-Galactopyranose and the two ring-isomeric D-galactofuranoses are oxidized, at high enzyme concentrations, only after their rate-limiting isomerization into the b-D-pyranose. The open-chained aldehyde form of D-galactose has been likewise excluded as substrate.3. D-Galactono-l,5-lactone has been shown by gas-liquid chromatography to be the immediate product of the enzymatic dehydrogenation of D-galactose and identified by combined gas-liquid chromatography -mass spectrometry of the isolated trimethylsilylated derivative.4. The primarily formed D-galactono-l,5-lactone rearranges non-enzymatically to the corresponding D-galactono-l,4-lactone by an intramolecular rearrangement without the participation of the open-chained D-galactonate. This rearrangement is strongly dependent on the pH-value, having a first-order rate constant k = 1.0 min-' at pH 6.8 under the conditions used. were determined at pH 6.8 under the conditions used, enabling the estimation of the apparent equilibrium constant for the reaction catalyzed by the enzyme6. Results analogous to those with D-galactose were obtained from investigations with the homeomorphic L-arabinose. The pyranose ring with the hydroxyl group at carbon-1 in the equatorial position is likewise the only substrate, and the immediate product, the 1,5-lactone, also rearranges subsequently to the corresponding 1,4-lactone, having a first-order rate constant k = 0.22 min-l at pH 6.8 under the conditions used. The ~-arabinono-1,5-lactone has also been identified by gas-liquid chromatography -mass spectrometry of the trimethylsilylated derivative.

Crystal Structure of Uronate Dehydrogenase from Agrobacterium tumefaciens

“…3). Similar non-enzymatic 1,4-lactone formation has been observed previously for D-galactose dehydrogenases (20). Because the rearrangement is an equilibrium reaction, a very small amount of D-galactaro-1,5-lactone exists in solution and explains why this form was observed in the ternary complex crystal structure.…”

NMR Spectroscopic Analysis of the First Two Steps of the Pentose-Phosphate Pathway Elucidates the Role of 6-Phosphogluconolactonase

“…␥-6-Phosphogluconolactone is formed subsequently by the rearrangement of the ␦-6-phosphogluconolactone. It had been established already that some ␦ lactones were spontaneously converted into the ␥ forms without participation of the open chain of the corresponding acids (20). Our kinetic study revealed that each 6-P-G-L is convertible into the other one, the conversion of the ␦-6-P-G-L into the ␥-6-P-G-L being characterized by a higher rate constant than the reverse conversion.…”

δ‐Galactonolactone: Synthesis, Isolation, and Comparative Structure and Stability Analysis of an Elusive Sugar Derivative

“…Table 4 lists the experimentally determined ratios and free enthalpies calculated by van't Hoff's equation for the δǞγ isomerization. It should be noted that the experimentally determined isomer distributions presented here cannot be compared directly with any previously published data, [3,11,41] since all of them were obtained in (buffered) aqueous solutions and, thus, include hydrolysis to the free aldonic acids, which is not observed in the dry DMSO medium employed in our study. [a] By integration of signals in 1 H NMR spectra.…”

“…10 h for 4a/b (i.e., seven halflives each), while 5a/b requires weeks to attain its equilibrium. Overall, the isomerization process in [D 6 ]DMSO is orders of magnitudes slower than the value of k ϭ 1 min Ϫ1 ϭ 1.67 ϫ 10 Ϫ2 s Ϫ1 observed for 6a in aqueous solution buffered at pH ϭ 6.8, [11] which suggests that, in aqueous solution, the isomerization is susceptible to the general acid/base catalysis that is also observed in the hydrolysis of the lactones and precludes the isolation of 6a from aqueous solution. [42] In addition, the relative rates of isomerization for the lactones 4a and 5a observed by us in [D 6 ]DMSO are opposite to those previously noted in the literature.…”