Basic residues play key roles in catalysis and NADP(+)-specificity in maize (Zea mays L.) photosynthetic NADP(+)-dependent malic enzyme.
C(4)-specific (photosynthetic) NADP(+)-dependent malic enzyme (NADP(+)-ME) has evolved from C(3)-malic enzymes and represents a unique and specialized form, as indicated by its particular kinetic and regulatory properties. In the present paper, we have characterized maize (Zea mays L.) photosynthetic NADP(+)-ME mutants in which conserved basic residues (lysine and arginine) were changed by site-directed mutagenesis. Kinetic characterization and oxaloacetate partition ratio of the NADP(+)-ME K255I (Lys-255-->Ile) mutant suggest that the mutated lysine residue is implicated in catalysis and substrate binding. Moreover, this residue could be acting as a base, accepting a proton in the malate oxidation step. At the same time, further characterization of the NADP(+)-ME R237L mutant indicates that Arg-237 is also a candidate for such role. These results suggest that both residues may play 'back-up' roles as proton acceptors. On the other hand, Lys-435 and/or Lys-436 are implicated in the coenzyme specificity (NADP(+) versus NAD(+)) of maize NADP(+)-ME by interacting with the 2'-phosphate group of the ribose ring. This is indicated by both the catalytic efficiency with NADP(+) or NAD(+), as well as by the reciprocal inhibition constants of the competitive inhibitors 2'-AMP and 5'-AMP, obtained when comparing the double mutant K435/6L (Lys-435/436-->Ile) with wild-type NADP(+)-ME. The results obtained in the present work indicate that the role of basic residues in maize photosynthetic NADP(+)-ME differs significantly with respect to its role in non-plant MEs, for which crystal structures have been resolved. Such differences are discussed on the basis of a predicted three-dimensional model of the enzyme.
Detarsio
Enrique
E
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Suipacha 531, Rosario, Sante Fe 2000, Argentina.
Andreo
Carlos S
CS
Drincovich
María F
MF
D000595:Amino Acid Sequence; D001120:Arginine; D002384:Catalysis; D017124:Conserved Sequence; D007700:Kinetics; D008239:Lysine; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010788:Photosynthesis / Q000502:physiology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology*
D011994:Recombinant Proteins; D001120:Arginine; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+); D008239:Lysine