Expression, purification, and characterization of recombinant nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase from Clostridium acetobutylicum.
Clostridium acetobutylicum gapN was cloned and expressed in Escherichia coli BL-21. The IPTG-induced nonphosphorylating NADP-dependent GAPDH (GAPN) has been purified about 34-fold from E. coli cells and its physical and kinetic properties were investigated. The purification method consisted of a rapid and straightforward procedure involving anion-exchange and hydroxyapatite chromatographies. The purified protein is an homotetrameric of 204kDa exhibiting absolute specificity for NADP. Chromatofocusing analysis showed the presence of only one acidic GAPN isoform with an acid isoelectric point of 4.2. The optimum pH of purified enzyme was 8.2. Studies on the effect of assay temperature on enzyme activity revealed an optimal value of about 65 degrees C with activation energy of 18KJmol(-1). The apparent K(m) values for NADP and D-glyceraldehyde-3-phosphate (D-G3P) or DL-G3P were estimated to be 0.200+/-0.05 and 0.545+/-0.1 mM, respectively. No inhibition was observed with L-D3P. The V(max) of the purified protein was estimated to be 78.8 U mg(-1). The Cl. acetobutylicum GAPN was markedly inhibited by sulfhydryl-modifying reagent iodoacetamide, these results suggest the participation of essential sulfhydryl groups in the catalytic activity.
Laboratoire de BBCM, Département de Biologie, Faculté des Sciences Aïn-Chock, Université Hassan-II, Km 8 route d'El Jadida, B.P. 5366 Mâarif, Casablanca, Morocco.
Abdelghani
A
Iddar
Federico
F
Valverde
Aurelio
A
Serrano
Abdelaziz
A
Soukri
D002384:Catalysis; D003013:Clostridium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009249:NADP / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D009249:NADP; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)