Multiple roles of arginine 181 in binding and catalysis in the NAD-malic enzyme from Ascaris suum.

The NAD-malic enzyme catalyzes the oxidative decarboxylation of l-malate. Structures of the enzyme indicate that arginine 181 (R181) is within hydrogen bonding distance of the 1-carboxylate of malate in the active site of the enzyme and interacts with the carboxamide side chain of the nicotinamide ring of NADH, but not with NAD+. Data suggested R181 might play a central role in binding and catalysis in malic enzyme, and it was thus changed to lysine and glutamine to probe its potential function. A nearly 100-fold increase in the Km for malate and a 30-fold increase in the Ki for oxalate, an analogue of the enolpyruvate intermediate, in the R181Q and R181K mutants are consistent with a role for R181 in binding substrates. The mutant enzymes also exhibit a >10-fold increase in KiNADH, but only a slight or no change in KNAD, consistent with rotation of the nicotinamide ring into the malate binding site upon reduction of NAD+ to NADH. The activity of the R181Q mutant can be rescued by ammonium ion likely by binding in the pocket vacated by the guanidinium group of R181. Results suggest 2 mol of ammonia bind per mole of active sites with a high-affinity KNH4 of 0.7 +/- 0.1 mM and a low-affinity KNH4 of approximately 420 mM. Occupancy of the high-affinity site, likely by NH4+, results in an increase in the affinity of malate, oxalate, and NADH (with no change in NAD affinity), consistent with the above-proposed roles for R181. The second molecule to bind is likely neutral NH3, and its binding increases V/Et approximately 20-fold. Primary deuterium and 13C isotope effects measured in the absence and presence of ammonium ion suggest R181Q predominantly affects the rate of the reaction by changing the rate of the precatalytic conformational change. The isotope effects do not change upon binding the second mole of ammonia in spite of the 20-fold increase in V/Et. Thus, the R181Q mutant enzyme exists as an equilibrium mixture between active and less active forms, and NH3 stabilizes the more active conformation of the enzyme.