pH Effects on the Activity and Regulation of the NAD Malic Enzyme.
The NAD malic enzyme shows a pH optimum of 6.7 when complexed to Mg(2+) and NAD(+) but shifts to 7.0 when the catalytically competent enzyme-substrate (E-S) complex forms upon binding malate(-2). This is characteristic of an induced conformational change. The slope of the V(max) or V(max)/K(m) profiles is steeper on the alkaline side of the pH optimum. The K(m) for malate increases markedly under alkaline conditions but is not greatly affected by pH values below the optimum. The loss of catalysis on the acidic side is due to protonation of a single residue, pK 5.9, most likely histidine. Photooxidation inactivation with methylene blue showed that a histidine is required for catalytic activity. The location of this residue at or near the active site is revealed by the protection against inactivation offered by malate. Three residues, excluding basic residues such as lysine (which have also been shown to be vital for catalytic activity, must be appropriately ionized for malate decarboxylation to proceed optimally. Two of these residues directly participate in the binding of substrates and are essential for the decarboxylation of malate. A pK of 7.6 was determined for the two residues required by the E-S complex to achieve an active state, this composite value representing both histidine and cysteine suggests that both have decisive roles in the operation of the enzyme. A major change in the enzyme takes place as protonation nears the pH optimum, this is recorded as a change in the enzyme's intrinsic affinity for malate (K(m pH6.7) = 9.2 millimolar, K(m pH7.7) = 28.3 millimolar). Similar changes in K(m) have been observed for the NAD malic enzyme as it shifts from dimer to tetramer. It is most likely that the third ionizable group (probably a cysteine) revealed by the V(max)/K(m) profile is needed for optimal activity and is involved in the association-dissociation behavior of the enzyme.
Willeford
K O
KO
Department of Biochemistry, University of California, Riverside, California 92521.
Wedding
R T
RT