Mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate.
The mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate has been probed using initial velocity studies, deuterium isotope effects, and isotope partitioning of the E:Mg:malate complex. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:NAD:Mg:malate complexes. Fumarate is a positive heterotropic effector of the NAD-malic enzyme at low concentrations (K act approximately 0.05 mM) and an inhibitor competitive against malate (Ki approximately 25 mM). The activation by fumarate results in a decrease in the Ki malate and an increase in V/K malate of about 2-fold, while the maximum velocity remains constant. Isotope partitioning studies of E:Mg:[14C]malate indicate that the presence of fumarate results in a decrease in the malate off-rate constant by about 2.2-fold. The deuterium isotope effects on V and V/K malate are both 1.6 +/- 0.1 in the absence of fumarate, while in the presence of 0.5 mM fumarate DV is 1.6 +/- 0.1 and D(V/K malate) is 1.1 +/- 0.1. These data are also consistent with a decrease in the off-rate for malate from E:NAD:Mg:malate, resulting in an increase in the forward commitment factor for malate and manifested as a lower value for D(V/K malate). There is a discrimination between active and activator sites for the binding of dicarboxylic acids, with the activator site preferring the extended configuration of 4-carbon dicarboxylic acids, while the active site prefers a configuration in which the 4-carboxyl is twisted out of the C1-C3 plane. The physiologic importance and regulatory properties of fumarate in the parasite are also discussed.
Lai
C J
CJ
Department of Biochemistry & Molecular Biology, Texas College of Osteopathic Medicine, Fort Worth 76107.
Harris
B G
BG
Cook
P F
PF
D000818:Animals; D017165:Ascaris suum / Q000201:enzymology*; D004789:Enzyme Activation; D005650:Fumarates / Q000378:metabolism*; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D013379:Substrate Specificity
D005650:Fumarates; D008293:Malates; D009243:NAD; D008291:Malate Dehydrogenase