Regulation of C4 photosynthesis: physical and kinetic properties of active (dithiol) and inactive (disulfide) NADP-malate dehydrogenase from Zea mays.
NADP-malate dehydrogenase was purified from leaves of Zea mays in the absence of thiol-reducing agents by (NH4)2SO4, polyethylene glycol, and pH fractionation followed by dye-ligand affinity chromatography and gel filtration. The purified enzyme is completely inactive (no activity detected between pH 6 and 9) but can be reactivated by thiol-reducing agents including dithiothreitol and thioredoxin. The active enzyme shows distinctly alkaline pH optima when assayed in either direction; Km values at pH 8.5 are oxaloacetate, 18 microM; malate, 24 mM; NADPH, 50 microM; and NADP, 45 microM. The reduction of oxaloacetate is inhibited by NADP (competitive with respect to NADPH, Ki = 50 microM). The molecular weight of the native inactive or active enzyme is 150,000 with subunits of Mr 38,000. Active enzyme is much more sensitive (greater than 50-fold) to heat denaturation than is the inactive enzyme and is irreversibly inactivated by N-ethylmaleimide whereas the inactive enzyme is insensitive to this reagent. The active and inactive forms of NADP-malate dehydrogenase are assumed to correspond to dithiol and disulfide forms of the enzyme, respectively. The relative coenzyme-binding affinities of inactive NADP-malate dehydrogenase differ by a factor of 10(2) from the binding affinities for active NADP-malate dehydrogenase and 10(4) for non-thiol-regulated NAD-specific malate dehydrogenase. It is proposed that the 100-fold change in differential binding of NADP and NADPH upon conversion of NADP-malate dehydrogenase to the disulfide form may sufficiently alter the equilibrium of the central enzyme-substrate complexes, and hence the catalytic efficiency of the enzyme, to explain the associated loss of activity.
D002244:Carbon / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D004220:Disulfides / Q000378:metabolism; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010788:Photosynthesis; D014050:Toluene / Q000031:analogs & derivatives / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology*
D004220:Disulfides; D014050:Toluene; D002244:Carbon; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+); C004848:dithiol