Glucose-6-phosphate dehydrogenase from Leuconosfoc rnesenferoides

Glucose-6-phosphate dehydrogenase (G6PD) from Leuconostoc mesenteroides is one of a small number of dehydrogenases that can utilize either NAD or NADP. The physiological roles of these two coenzymes are quite different and the enzymes that utilize them are generally specific for one or the other coenzyme. An essential function of G6PDs in animals, for example, is to generate NADPH for reductive biosyntheses. Although these enzymes can utilize NAD+, they d o so only at non-physiological NAD' concentrations [ 1, 21. Presumably the coenzyme-binding sites of dehydrogenases have evolved so that they can distinguish between NAD and NADP. L. mesenteroides is a facultactively anaerobic micro-organism which metabolizes glucose to generate lactic acid, ethanol and CO, [3]. The NADH generated in the reaction catalysed by G6PD is utilized in the reductive steps of this heterolactic fermentation; when NADPH is produced by G6PD it is used for the biosynthesis of fatty acids [4]. Thus, G6PD plays an essential, amphibolic role in the metabolism of L. mesenteroides. Two questions that have motivated our studies of L. mesenteroides G6PD are: what protein structural features enable this enzyme to bind both NAD+ and NADP' to the same site? and how are the catalytic activities with NAD+ and NADP' regulated? Unlike glutamate dehydrogenase from animal tissues, another dehydrogenase that utilizes either NAD+ or NADP+, L. mesenteroides G6PD is a relatively simple protein consisting of two identical subunits of molecular mass 54 800 Da, with no propensity to form larger aggregates [ 5 , 61. Kinetic studies revealed that the enzyme obeys Michaelis-Menten kinetics with no evidence of cooperative kinetics. The K , for NAD+ is nearly 20 times greater than the K , for NADP', whereas k,,,, for the NAD-linked reaction is nearly twice k,,,, for the reaction with NADP+ (Table 1) [7, 81. Kinetic and binding studies showed that NAD' and NADP+ bind to the same site on the enzyme [7,9], although it is clear that their modes of binding differ. The steady-state kinetic mechanisms of the NADand NADP-linked reac-