title: Biochemical characterization of glyceraldehyde-3-phosphate dehydrogenase from Thermococcus kodakarensis KOD1.
abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an essential role in glycolysis by catalyzing the conversion of D-glyceraldehyde 3-phosphate (D-G3P) to 1,3-diphosphoglycerate using NAD(+) as a cofactor. In this report, the GAPDH gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (GAPDH-tk) was cloned and the protein was purified to homogeneity. GAPDH-tk exists as a homotetramer with a native molecular mass of 145 kDa; the subunit molecular mass was 37 kDa. GAPDH-tk is a thermostable protein with a half-life of 5 h at 80-90°C. The apparent K (m) values for NAD(+) and D-G3P were 77.8 ± 7.5 μM and 49.3 ± 3.0 μM, respectively, with V (max) values of 45.1 ± 0.8 U/mg and 59.6 ± 1.3 U/mg, respectively. Transmission electron microscopy (TEM) and image processing confirmed that GAPDH-tk has a tetrameric structure. Interestingly, GAPDH-tk migrates as high molecular mass forms (~232 kDa and ~669 kDa) in response to oxidative stress.
authors.affiliation: College of Plant Sciences, Jilin University, Changchun, 130-062, China.
authors.forename: Baolei
authors.initials: B
authors.lastname: Jia
authors.forename: Le Thuy
authors.initials: le T
authors.lastname: Linh
authors.forename: Sangmin
authors.initials: S
authors.lastname: Lee
authors.forename: Bang Phuong
authors.initials: BP
authors.lastname: Pham
authors.forename: Jinliang
authors.initials: J
authors.lastname: Liu
authors.forename: Hongyu
authors.initials: H
authors.lastname: Pan
authors.forename: Shihong
authors.initials: S
authors.lastname: Zhang
authors.forename: Gang-Won
authors.initials: GW
authors.lastname: Cheong
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004163:Diphosphoglyceric Acids / Q000378:metabolism; D004795:Enzyme Stability; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006207:Half-Life; D006358:Hot Temperature; D007700:Kinetics; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009243:NAD / Q000378:metabolism; D018384:Oxidative Stress; D020836:Protein Structure, Quaternary; D013329:Structure-Activity Relationship; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; D004163:Diphosphoglyceric Acids; D009243:NAD; D005986:Glyceraldehyde 3-Phosphate; C015891:glycerate 1,3-biphosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases