Kinetic studies of the regulation of mitochondrial malate dehydrogenase by citrate

Abstract

Mitochondrial malate dehydrogenase shows a complex regulation pattern in the presence of citrate. Previously published results indicate that this enzyme is activated by citrate in the NAD(+)----NADH direction and inhibited in the opposite direction. Moreover, high concentrations of L-malate or oxaloacetate produce deviations from the Michaelis-Menten behaviour. Results reported in this paper clearly show that citrate both activates and inhibits mitochondrial malate dehydrogenase in the same direction (NAD(+)----NADH), and in the same reaction medium, depending on substrate concentration. This surprising effect has made it necessary to propose a new kinetic mechanism that extends those previously suggested and allows us to explain both the citrate effect (activating or inhibitory) and the effect of high concentrations of L-malate and oxaloacetate.

D‐2‐Hydroxy‐4‐Methylvalerate Dehydrogenase from Lactobacillus Delbrueckii Subsp. Bulgaricus— I. Kinetic Mechanism and pH Dependence of Kinetic Parameters, Coenzyme Binding and Substrate Inhibition

“…The K,,, value for the 2-oxoacid is comparable to the reported values for members of the L-2-hydroxyacid dehydrogenase fam- ily [18,22,231 but much smaller than those for other members of D-2-hydroxyacid dehydrogenase such as D-laCtate dehydrogenases from Lactobacillus pentosus [24] and Lactobacillus helveticus [25]. We also note the k,,, value of ~-2-hydroxy-4-methylvalerate dehydrogenase with O(Me)vl is much lower than that of D-lactate dehydrogenase with pyruvate [25], in spite of the two enzymes catalysing a similar chemical reaction and having similar primary structures [4].…”

A mathematical model of the mitochondrial NADH shuttles and anaplerosis in the pancreatic β-cell

“…These constraints, of course, demand that experimental data on the kinetics of the enzyme agree with the assumptions. This is indeed the case, as may be verified from the values in Table 10, which were inferred from the study of Gelpí et al (34). Some properties of MDHm were not considered here; first, the citrate-dependent substrate activation by malate, which occurs only at NAD concentrations higher than those assumed in our study; and second, the substrate inhibition by oxaloacetate, which occurs only at much higher oxaloacetate concentrations than those assumed here (9,34).…”

“…In contrast to the cytosolic isozyme, MDHm is allosterically regulated in a peculiar manner. Experiments (34) indicate that the allosteric effector citrate may act as an inhibitor or an activator for the malate oxidation reaction, depending on the NAD concentration. A detailed analysis (34) showed that citrate increases the effective limiting rate V MDHm while at the same time also increasing the half-activation points for malate and NAD.…”

“…Experiments (34) indicate that the allosteric effector citrate may act as an inhibitor or an activator for the malate oxidation reaction, depending on the NAD concentration. A detailed analysis (34) showed that citrate increases the effective limiting rate V MDHm while at the same time also increasing the half-activation points for malate and NAD. In the reverse direction, citrate increases the half-activation point for NADH while leaving the half-activation point for oxaloacetate unchanged, as well as the backward limiting rate.…”

Enzymatic pattern processing

“…The reaction was monitored by spectroscopic detection of NADH at 339 nm. MDH has been one of the most intensively studied enzymes so far as response to milieu conditions and kinetic mechanisms are concerned (Wolfe and Raval 1970;DuVal et al 1985;Gelpí et al 1992). However, our purpose here is not to investigate the mechanism but rather to utilize the phenomenology.…”