title: Enzyme Architecture: A Startling Role for Asn270 in Glycerol 3-Phosphate Dehydrogenase-Catalyzed Hydride Transfer.
abstract: The side chains of R269 and N270 interact with the phosphodianion of dihydroxyacetone phosphate (DHAP) bound to glycerol 3-phosphate dehydrogenase (GPDH). The R269A, N270A, and R269A/N270A mutations of GPDH result in 9.1, 5.6, and 11.5 kcal/mol destabilization, respectively, of the transition state for GPDH-catalyzed reduction of DHAP by the reduced form of nicotinamide adenine dinucleotide. The N270A mutation results in a 7.7 kcal/mol decrease in the intrinsic phosphodianion binding energy, which is larger than the 5.6 kcal/mol effect of the mutation on the stability of the transition state for reduction of DHAP; a 2.2 kcal/mol stabilization of the transition state for unactivated hydride transfer to the truncated substrate glycolaldehyde (GA); and a change in the effect of phosphite dianion on GPDH-catalyzed reduction of GA, from strongly activating to inhibiting. The N270A mutation breaks the network of hydrogen bonding side chains, Asn270, Thr264, Asn205, Lys204, Asp260, and Lys120, which connect the dianion activation and catalytic sites of GPDH. We propose that this disruption dramatically alters the performance of GPDH at these sites.
authors.affiliation: Department of Chemistry, University at Buffalo , Buffalo, New York 14260-3000, United States.
authors.forename: Archie C
authors.initials: AC
authors.lastname: Reyes
authors.affiliation: Department of Chemistry, University at Buffalo , Buffalo, New York 14260-3000, United States.
authors.forename: Tina L
authors.initials: TL
authors.lastname: Amyes
authors.affiliation: Department of Chemistry, University at Buffalo , Buffalo, New York 14260-3000, United States.
authors.forename: John P
authors.initials: JP
authors.lastname: Richard
mesh_terms: D001216:Asparagine / Q000502:physiology*; D001880:Boranes / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain / Q000502:physiology*; D018360:Crystallography, X-Ray; D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000201:enzymology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D001880:Boranes; D001216:Asparagine; D005993:Glycerolphosphate Dehydrogenase