title: Unfolding and trypsin inactivation studies reveal a conformation drift of glucose-6-phosphate dehydrogenase upon binding of NADP.
abstract: Binding of NADP to glucose-6-phosphate dehydrogenase (G6PD) from Dicentrarchus labrax liver has stabilized its native structure against thermal inactivation, guanidine hydrochloride unfolding and inactivation by tryptic digestion. The time-course of G6PD inactivation by guanidine hydrochloride in the presence of NADP has provided experimental evidence in favor of a conformational drift upon NADP binding to the bass enzyme. Based on the inactivation patterns obtained when the enzyme was treated with guanidine hydrochloride and trypsin, it is proposed that the enzyme conformation induced upon NADP binding is in slow equilibrium with the conformation stabilized in the absence of NADP. FPLC studies have shown that micromolar concentrations of NADP induced oligomerization of G6PD. In addition, the different K0.5 values of NADP binding to the enzyme, ranging from 1-2 microM (from trypsin inactivation) to 90 microM (from titration of the intrinsic fluorescence), suggest a step-wise binding of NADP to the oligomer, with negative cooperativity in the saturation process.
authors.affiliation: Departamento de Bioquímica y Biología Molecular IV, Universidad Complutense de Madrid, Facultad de Veterinaria, Spain.
authors.forename: J M
authors.initials: JM
authors.lastname: Bautista
authors.forename: J M
authors.initials: JM
authors.lastname: Fuentes
authors.forename: A
authors.initials: A
authors.lastname: Diez
authors.forename: C
authors.initials: C
authors.lastname: Gutiérrez-Merino
authors.forename: G
authors.initials: G
authors.lastname: Soler
mesh_terms: D000818:Animals; D001492:Bass; D004789:Enzyme Activation / Q000187:drug effects; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006358:Hot Temperature; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014357:Trypsin
chemical_list: D006146:Guanidines; D009249:NADP; D005954:Glucosephosphate Dehydrogenase; D014357:Trypsin; D019791:Guanidine