acid cycle, malate dehydrogenase is responsible for catalyzing the regeneration of oxaloacetate This reaction occurs through the oxidation of hydroxyl group on malate and reduction of NAD+. The mechanism of the transfer of the hydride ion to NAD+ is carried out in a similar mechanism seen in lactate dehydrogenase and alcohol dehydrogenase. The ΔG'° of malate dehydrogenase is +29.7 kJ/mol and the ΔG (in the cell) is 0 kJ/mol. Other pathways Malate dehydrogenase is also involved in gluconeogenesis, the synthesis of glucose from smaller molecules. Pyruvate in the mitochondria is acted upon by pyruvate carboxylase to form oxaloacetate, a citric acid cycle intermediate. In order to get the oxaloacetate out of the mitochondria, malate dehydrogenase reduces it to malate, and it then traverses the inner mitochondrial membrane. Once in the cytosol, the malate is oxidized back to oxaloacetate by cytosolic malate dehydrogenase. Finally, phosphoenolpyruvate carboxykinase (PEPCK) converts oxaloacetate to phosphoenolpyruvate (PEP). Kinetics Kinetic studies show that malate dehydrogenase enzymatic activity is ordered. The cofactor NAD+/NADH is bound to the enzyme before the substrate. The Km value for malate, i.e., the concentration at which the enzyme activity is half-maximal, is 2 mM. The Kcat value is 259.2 s−1. Effect of pH on catalytic activity Additionally, pH levels control specificity of substrate binding by malate dehydrogenase due to proton transfer in the catalytic mechanism. A histidine moiety with a pK value of 7.5 has been suggested to play a role in the pH-dependency of the enzyme. Studies have indicated that the binding of the enol form oxaloacetate with the malate dehydrogenase:NADH complex forms much more rapidly at higher pH values. Additionally, L-malate binding to malate dehydrogenase is promoted at alkaline conditions. Consequently, the non-protonated form malate dehydrogenase binds preferentially to L-malate and the enol form of oxaloacetate. In contrast, D-malate, hydroxymalonate, and the keto form of oxaloacetate have been found to bind exclusively to the protonated form of the enzyme. Specifically, when the histidine is protonated, the His residue can form a hydrogen bond with the substrate's carbonyl oxygen, which shifts electron density away from the oxygen and makes it more susceptible to nucleophilic attack by hydride. This promotes the binding of malate dehydrogenase to these substrates. As a result, at lower pH values malate dehydrogenase binds preferentially to D-malate, hydroxymalonate, and keto-oxaloacetate. Allosteric regulation Because malate dehydrogenase is closely tied to the citric acid cycle, studies have proposed and experimentally demonstrated