Enzymatic degradation of cyclic 2,3-diphosphoglycerate to 2,3-diphosphoglycerate in Methanobacterium thermoautotrophicum.
2,3-Diphosphoglycerate (2,3-DPG) has been found to be the product of the enzymatic degradation of cyclic 2,3-diphosphoglycerate (cDPG) in the archaebacterium Methanobacterium thermoautotrophicum delta H. Although 2,3-DPG has not previously been detected as a major soluble component of M. thermoautotrophicum, large pools accumulated at an incubation temperature of 50 degrees C (below the optimum growth temperature of 62 degrees C). Under these conditions, cellular activity was significantly decreased; a return of the culture to the optimum growth temperature restored the 2,3-DPG pool back to original low levels and caused steady-state cDPG levels to increase again. While 13CO2-pulse/12CO2-chase experiments at 50 degrees C showed that the cDPG turned over, the appearance of 2,3-DPG at NMR-visible concentrations required at least 10 h. Production of 2,3-DPG in vivo was prevented by exposure of the cells to O2. The enzyme responsible for this hydrolysis of cDPG was purified by affinity chromatography and appears to be a 33-kDa protein. Activity was detected in the presence of oxygen and was enhanced by a solution of 1 M KCl, 25 mM MgCl2, and dithiothreitol. Both Km and Vmax have been determined at 37 degrees C; kinetics also indicate that in vitro the product, 2,3-DPG, is an inhibitor of cDPG hydrolysis. These findings are discussed in view of a proposed role for cDPG in methanogens.
Sastry
M V
MV
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02167.
Robertson
D E
DE
Moynihan
J A
JA
Roberts
M F
MF
D019794:2,3-Diphosphoglycerate; D002846:Chromatography, Affinity; D004163:Diphosphoglyceric Acids / Q000378:metabolism*; D006867:Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D017014:Methanobacterium / Q000201:enzymology*; D010100:Oxygen / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D013053:Spectrophotometry; D013696:Temperature
D004163:Diphosphoglyceric Acids; D019794:2,3-Diphosphoglycerate; D011189:Potassium Chloride; C039905:cyclic-2,3-diphosphoglycerate; D006867:Hydrolases; D010100:Oxygen