Evidence for the Existence of Two Essential and Proximal Cysteinyl Residues in NADP-Malic Enzyme from Maize Leaves

Abstract

Incubation of maize (Zea mays) leaf NADP-malic enzyme with monofunctional and bifunctional N-substituted maleimides results in an irreversible inactivation of the enzyme. Inactivation by the monofunctional reagents, N-ethylmaleimide (NEM) and N-phenylmaleimide, followed pseudo-first-order kinetics. The maximum inactivation rate constant for phenylmaleimide was 10-fold higher than that for NEM, suggesting a possible hydrophobic microenvironment of the residue(s) involved in the modification of the enzyme. In contrast, the inactivation kinetics with the bifunctional maleimides, ortho-, meta-, and para-phenylenebismaleimide, were biphasic, probably due to different reactivities of the groups reacting with the two heads of these bifunctional reagents, with a possible cross-linking of two sulfhydryl groups. plants such as maize. The enzyme plays a key role in C4 photosynthetic metabolism because it generates reducing power and CO2 in the bundle sheath chloroplasts where Rubisco and the Calvin cycle operate (7,8).Thiol groups play a key role in the activity of the enzyme obtained from different sources. The importance of sulfhydryl residues in pigeon liver malic enzyme has been investigated extensively (10). Studies of the inactivation of this enzyme with reagents selective for sulfhydryl groups, e.g. 5,5'-dithiobis(2-nitrobenzoic acid) or NEM2, confirmed the presence of one sulfhydryl group near each substrate site on the enzyme tetramer (24). In contrast, evidence for the presence of two essential sulfhydryl residues per monomer of the enzyme from maize leaves has been recently reported (4).To obtain further information concerning the function and microenvironment of the active site thiol group(s), the reaction of NADP-malic enzyme from maize leaves with different N-substituted maleimides was studied. Bifunctional maleimides were used to obtain further evidence about the existence of two proximal sulfhydryl groups near the NADP-binding site of the C4 enzyme (4). Several isomers of phenylenebismaleimide with different estimated cross-linking distances, as well as maleimides that differ in hydrophobic properties, were all found to modify the enzyme in an irreversible fashion.NADP-dependent malic enzyme (L-malate: NADP oxidoreductase/decarboxylase; EC 1.1.1.40) acts in a wide range of metabolic pathways in both animals and plants. The enzyme catalyzes the reversible oxidative decarboxylation of L-malate in the presence of NADP and a bivalent metal ion to produce pyruvate, NADPH, and CO2. In animals, cytosolic malic enzyme generates reducing power for the biosynthesis of fatty acids (9, 28). In plants, two forms of this enzyme are known to occur and have important metabolic roles. The cytosolic form is thought to participate in the regulation of intracellular pH (3,20) and/or in the provision of reducing power that can be used in processes requiring NADPH (8).The chloroplast stromal form is found specifically in the bundle sheath chloroplasts of NADP-malic enzyme type C4

Chimeric Structure of the NAD(P)+- and NADP+-dependent Malic Enzymes ofRhizobium (Sinorhizobium) meliloti

“…The two lysine residues in region B are believed to be required for NAD(P) ϩ binding (36). Region C contains a conserved cysteine residue thought to be located at or near the active site of the eukaryotic enzymes (40,41) and may be responsible for coordinating the binding of the substrate L-malate or the divalent metal ion (2). Region D represents a large block of 13 amino acid residues that are absolutely conserved in the prokaryotic malic enzymes.…”

Characterization of a Bean (Phaseolus vulgaris L.) Malic‐enzyme Gene

“…The existence of a sulfhydryl group at or near the NADP-binding site has been demonstrated [39]. Additional experiments provided evidence for two proximal active-site thiol groups [40]. Protection by NADP of enzyme inactivation imposed by addition of maleimides indicates that both residues are at or near the NADP-binding site(s).…”