D-Mannitol dehydrogenase from Absidia glauca. Steady-state kinetic properties and the inhibitory role of mannitol 1-phosphate

Abstract

Steady-state kinetic studies including initial velocity for mannitol oxidation and fructose reduction and product inhibition for mannitol oxidation using fructose and reduced nicotinamide adenine dinucleotide (NADH) are in accord with a reaction mechanism best described as ordered Bi-Bi with NAD+ and NADH designated as the first substrate, last product, respectively at pH 8.8. All replots of slopes and intercepts from product inhibition studies were linear. Dead-end inhibition studies using mannitol 1-phosphate gave slope-parabolic, intercept-linear noncompetitive inhibition for both NAD+ and mannitol as substrates. The dead-end inhibitor is capable of binding multiply to the E, EA, and EQ forms of the enzyme to an extent that is controlled by the concentration of substrates. The EQ complex is inferred to undergo a conformational change, E'Q equilibrium EQ, since (V1/E1) greater than (KiqV2)/(KqE1), and no evidence for dead-end complex formation with NADH can be adduced. This is interpreted to mean that the release of fructose from the central complex is faster than the isomerization of the E-NADH complex. When mannitol is saturating, the noncompetitive inhibition against NAD+, as the variable substrate, becomes parabolic uncompetitive. A replot of the slopes of the parabola against mannitol 1-phosphate remains concave upward. This situation could arise if the conformational change we infer in the EQ complex opens up additional sites on the protein which can interact with the dead-end inhibitor.

Kinetic Study of the Catalytic Mechanism of Mannitol Dehydrogenase from Pseudomonas fluorescens

“…Another clear difference between MDH and SDH is the fact that the ternary E-NADH-fructose complex (39), the K iNADH value for MDH has been raised to a point where it equals the physiological concentration of the nucleotide which means that the extent of product inhibition of MDH by NADH will be low under these conditions. Interestingly, the analysis of the kinetic data published for MDH from A. glauca (7) leads to a conclusion similar to that outlined for MDH from P. fluorescens. The contribution of the K iNAD /K iNADH ratio (2.55) to the external equilibrium constant is approximately 38% (based on the log fraction), with the remaining 62% being contributed by the (k catr /K RO )/(k cato /K ROH ) ratio.…”

“…The initial step in the catabolism of D-mannitol involves oxidations at the C-1 (2, 3) and C-2 (4-6) positions, catalyzed by NAD(P)-dependent dehydrogenases. The reaction catalyzed by mannitol 2-dehydrogenase (MDH, 1 EC 1.1.1.67) is At present, there is little known about the structure-function relationships of MDH, and the kinetic and chemical mechanisms of MDH have not been studied, except for an early kinetic study with the MDH from the fungus Absidia glauca (7). The primary structures of three microbial MDHs from Pseudomonas fluorescens (8), Rhodobacter sphaeroides (9), and Rhodobacter capsulatus (GenBank accession number gi|3128349) have been determined together with those of hypothetical proteins in Saccharomyces cereVisiae and Escherichia coli.…”

Detection of Dye-linkedD-Mannitol Dehydrogenase Activity inAcetobacter xylinumKU-1

“…One unit of enzyme activity was defined as the amount of enzyme that reduced 1.0 tlmol of DCIP per min under the conditions used. NAD(P) + D-mannitol dehydrogenase activities were measured by the procedure of McGuinness et al 7 ) The assay mixture (1 ml) contained McIlvaine buffer, pH 4.6 (0.54ml), IOmM NAD(P)+ (0.1 ml), 0.5 M o-mannitol (0.2 ml), and deionized water (0.1 ml). The reaction was started by addition of enzyme solution (0.06 ml), and done at 30°C.…”