Lysine residues play key roles in catalysis and NADP-specificity in maize photosynthetic NADP-malic enzyme

DETARSIO, E.; ANDREO, CS; MARIA FABIANA DRINCOVICH

Biochemical Journal

Biochemical Society, Portland Press

Año: 2004 vol. 382 p. 1025 - 1030

0264-6021

C4-specific (photosynthetic) NADP-malic enzyme (NADP-ME) has evolved from C3-malic enzymes to represent a unique and specialized form of enzyme as indicated by its particular kinetic and regulatory properties. In the present work, we characterize maize photosynthetic NADP-ME mutants in conserved basic residues. Kinetic characterization and oxaloacetate partition ratio of NADP-ME K255I mutant suggest that the mutated lysine residue is implicated in catalysis and substrate binding, being a likely candidate for acting as the general base that accepts a proton from malate in the oxidation step. Nevertheless, further characterization of the NADP-ME R237L mutant indicates that arginine at position 237 is also a candidate for such role. In this way, it is suggested that both residues may play ´´back-up´´ roles for accepting the proton. On the other hand, lysine at position 435 and/or 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 suggested 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, that presents the mutant K435/6L with respect to the wild-type NADP-ME. The results obtained in the present work indicate that the role of lysine residues in maize photosynthetic NADP-ME differs significantly with respect to the role in non-plant MEs, which crystal structures have been resolved. Such differences are discussed on basis of a predicted three dimensional model of the enzyme