NADP-malate dehydrogenase from leaves of Zea mays: purification and physical, chemical, and kinetic properties.
NADP-malate dehydrogenase (NADP-MDH) from leaves of Zea mays has been purified and has a specific activity of 600-1000 mumol/min/mg protein. The native, inactive form of the enzyme is an 87.4-kDa, dimeric protein with a sedimentation coefficient of 5.5 S and a Stokes' radius of 3.62 nm. Isofocus analysis reveals the native enzyme preparation to contain two proteins of pI 4.88 and 4.90. The uv-visible absorbance spectrum reveals no chromophores on the protein. The inactive form of the enzyme contains three thiols and three disulfides per subunit. 2-Mercaptoethanol can reduce two of the three subunit disulfides without concomitant activation of the enzyme. Treating the enzyme with dithiothreitol reduces all three subunit disulfides and fully activates the enzyme. These results show that NADP-MDH activation is dependent on the reduction of a critical disulfide bond. The enzyme can use both NADPH and NADH for oxaloacetate (OAA) reduction and NADP and NAD for malate oxidation at the following measured specific activities (eu/mg protein) at pH 8.5 in Tris buffer: NADPH plus OAA (690), NADH plus OAA (260), NADP plus malate (82), and NAD plus malate (37). These activities vary as a function of pH and buffer composition. Km values for the substrate pairs are NADPH (24 microM) plus OAA (56 microM); NADH (0.83 mM) plus OAA (61 microM); NADP (73 microM) plus malate (32 mM); and NAD (0.80 mM) plus malate (29 mM). The enzyme shows allosteric kinetics for NADP with a Hill number of 1.56. The enzyme is substrate-inhibited by malate for both NADP- and NAD-dependent activities.
Kagawa
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Biochemistry Department, University of Missouri, Columbia 65211.
Bruno
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PL
D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D002850:Chromatography, Gel; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D010944:Plants / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000032:analysis; D003313:Zea mays
D000596:Amino Acids; D004220:Disulfides; D013438:Sulfhydryl Compounds; D009243:NAD; D009249:NADP; D008623:Mercaptoethanol; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)