Modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with the 2',3'-dialdehyde derivative of NADP+ (oNADP+).
1987; Elsevier BV; Volume: 262; Issue: 3 Linguagem: Inglês
10.1016/s0021-9258(19)75775-5
ISSN1083-351X
Autores Tópico(s)Metabolism and Genetic Disorders
ResumoGlucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is irreversibly inactivated by the 2,3'-dialdehyde of NADP+ (oNADP+) in the absence of substrate.The inactivation is first order with respect to NADP+ concentration and follows saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inhibitor followed by covalent modification (KI = 1.8 mM).NADP+ and NAD+ protect the enzyme from inactivation by oNADP+.The pK of inactivation is 8.1.oNADP+ is an effective coenzyme in assays of glucose-6-phosphate dehydrogenase Kinetic evidence and binding studies with ['"C] oNADP+ indicate that one molecule of oNADP+ binds per subunit of glucose-6-phosphate dehydrogenase when the enzyme is completely inactivated.The interaction between oNADP+ and the enzyme does not generate a Schiff's base, or a conjugated Schiff's base, but the data are consistent with the formation of a dihydroxymorpholino derivative.( K , = 200 jkM).Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides catalyzes the oxidation of D-glucopyranose 6-phos- phate to D-glucono-6-lactone-6-phosphate by NADP+ or NAD'.The enzyme was first isolated and its dual nucleotide specificity demonstrated by DeMoss et al. (1, 2).Kemp and Rose (3) showed that both NAD' and NADP' are utilized by L. mesenteroides glucose-6-phosphate dehydrogenase under physiological conditions and that the two reduced coenzyme products serve different metabolic processes in uiuo: NADPH is utilized in biosynthetic reactions and NADH is used in ATP-generating reactions in the production of ethanol and lactate.It is of interest to decipher the structural and chemical basis of the dual nucleotide specificity of this enzyme by identifying aspects of its active site structure that may relate to its ability to distinguish between the coenzymes.L. mesenteroides glucose-6-phosphate dehydrogenase consists of identical subunits of molecular weight 55,000 (4).Equilibrium dialysis experiments with NADP+ established that two independent binding sites exist per dimer; the Kd values for NAD' and NADP' were determined to be 7.2 mM and 6.5 PM, respectively ( 5 ) .The dual nucleotide specificity
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