Pre-steady-state kinetics reveal a slow isomerization of the enzyme-NAD complex in the NAD-malic enzyme reaction.
Stopped-flow experiments obtained in the pre-steady-state time scale of the NAD-malic enzyme reaction exhibit a lag prior to the attainment of steady state. Previous results from isotope effect studies in which the deuterium isotope effect on Vmax decreases to a value of 1 at low pH have been interpreted as suggesting a slow release of NADH [Kiick, D. M., Harris, B. G., & Cook, P. F. (1986) Biochemistry 25, 227-236]. The latter, however, requires a burst in the pre-steady-state time course, and thus the previous data have been reinterpreted in view of the observed lag. Preincubation with NAD and/or Mg increases the lag rate, with the latter having the greater effect, while preincubation with Mg and malate (or a malate analog) eliminates the lag. Data suggest a slow isomerization of E:NAD that is increased by addition of malate prior to NAD in the presence of Mg. The lag is also eliminated at low pH as a result of the overall rate being limited by the isomerization; that is, the isomerization is pH-dependent. Fumarate, an activator of the NAD-malic enzyme, when preincubated with enzyme also eliminates the lag, suggesting that the activator preferentially binds the isomerized form of the enzyme or increases the isomerization rate, or both. Stopped-flow data are corroborated by circular dichroism experiments. The unliganded enzyme is approximately 50% alpha-helix on the basis of secondary structural analysis. Binding of NAD and Mg exhibits a substantial change, with a further change observed upon binding the malate analog tartronate.
Rajapaksa
R
R
Department of Biochemistry and Molecular Biology, Texas College of Osteopathic Medicine, Fort Worth 76107.
Abu-Soud
H
H
Raushel
F M
FM
Harris
B G
BG
Cook
P F
PF
D000818:Animals; D017165:Ascaris suum / Q000201:enzymology; D002942:Circular Dichroism; D007536:Isomerism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D008962:Models, Theoretical; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors
D009243:NAD; D008291:Malate Dehydrogenase; D008274:Magnesium