First mechanism: First the proton is transferred from oxygen of malate to the nitrogen of the imidazole ring of the His-177 in the malate dehydrogenase (MDH). In a separate step, a hydride anion is transferred from the carbon of malate to the carbon of the nicotinamide ring of NAD to produce NADH. The initial step, the proton transfer, encounters an energy barrior of & kcal/mol and the next step, the hydride transfer, encounters an energy barrior of 15kcal/mol, which suggests the hydride transfer is the rate limiting step.

Alternate Mechanism: The alternate pathway is just the reverse of the first. The hydride transfers precedes the proton transfer. In this pathway the hydride transfer encounters an energy barrior of 54 kcal/mol. Because this is about 40 kcal/mole higher. This mechanism is not generally thought to be the pathway malate dehydrogenase uses.

Binding of Malate: The carboxylate groups at either end of malate are locked in place by H-bonds of arginine. Four salt bridges are formed between malate's oxygens and the nitrogens of MDH's Arg-153 and Arg-81.

Conformational change: As the enzyme-substrate complex moves to the proton transition state the imidazole ring rotates upward, leaving only 2.6 angstroms between malate's oxygen and the nitrogen of MDH's His 177. Malates oxygen shifts to within 2.56 angstroms of the nitrogen of MDH's Arg-87. The nitrogen of Asn-119 also moves to 2.9 angstoms of the oxygen. His-177 and Asp-150 move closer to each other.

Proton/Charge transfer: A charge change occurs on malate's oxygen which is stabilized by nitrogens of Arg-87 and Asn-119. In the transition state, little charge is transferred to the imidazole ring of His-177; however, a large charge change in the carbon of His-177 (-0.09e to -0.02e) occurs. After the transfer, +0.97e of charge is transferred to His-177.

Hydride transition state: Malate's carbon and the carbon of the nicotinamide ring move from 3.25 angstroms to 2.78 angstroms of each other while the oxygen of malate and nitrogens of Arg-87 and Asn-119 move away from each other. An energy transfer of -1e occurs when two electrons transfer with the proton to the nicotinamide ring of NAD. Malate's oxygen loses the negative charge accumulated after the proton transfer and the nitrogen atoms of Arg-87 and Asn-119 relax back to their original positions. The nicotinamide aquired-0.86e of charge at the hydride transition state, which it is distributed relatively uniformly.

Oxaloacetate State: The two H-bonds between malate and Arg-87 and the distance between malate's oxygen and the nitrogen of Asn-119 increases. The oxygen's final charge is now equal to the original charge (-0.42e) in the original malate state.

Energy: Because the pK_a of imidazole is 6.04, and the pK_a of the hydroxyl group is 13.0, the free energy calculated (free energy = -2.3RT[pK_a(imidazole) - pK_a(malate)] ) for the free energy of a proton transfer between malate and imidazole in aqueous solution is 9.6 kcal/mol.

INTRODUCTION

STRUCTURE

MECHANISM

REGULATION

REFERENCES