Biochemical characterization of glyceraldehyde-3-phosphate dehydrogenase from Thermococcus kodakarensis KOD1

초록

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 80-90A degrees C. The apparent K (m) values for NAD(+) and d-G3P were 77.8 +/- A 7.5 mu M and 49.3 +/- A 3.0 mu M, respectively, with V (max) values of 45.1 +/- A 0.8 U/mg and 59.6 +/- A 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 (similar to 232 kDa and similar to 669 kDa) in response to oxidative stress.

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