Purification of recombinant non-phosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from...

Streprococcus pyogenes gapN was cloned and expressed by functional complementation of the Escherichia gap mutant W3CG. The IPTG-induced NADP non-phosphorylating GAPDH (GAPN) has been purified about 75.4 fold from E. coli cells, using a procedure involving conventional ammonium sulfate fractionation, anion-exchange chromatography, hydrophobic chromatography and hydroxyapatite chromatography. The purified protein was characterised: it's an homotetrameric structure with a native molecular mass of 224 kDa, have an acid pI of 4.9 and optimum pH of 8.5. Studies on the effect of assay temperature on enzyme activity revealed an optimal value of about 60 degrees C with activation energy of 51 KJ mole(-1). The apparent Km values for NADP and D-G3P or DL-G3P were estimated to be 0.385 +/- 0.05 and 0.666 +/- 0.1 mM, respectively and the Vmax of the purified protein was estimated to be 162.5 U mg(-1). The S. pyogenes GAPN was markedly inhibited by sulfydryl-modifying reagent iodoacetamide, these results suggest the participation of essential sulfydryl groups in the catalytic activity.