Human brain 6-phosphogluconate dehydrogenase: purification and kinetic properties.
6-Phosphogluconate dehydrogenase has been purified from human brain to a specific activity of 22.8 U/mg protein. The molecular weight was 90,000. At low ionic strengths enzyme activity increased, due to an increase in Vmax and a decrease in Km for 6-phosphogluconate, and activity subsequently decreased as the ionic strength was increased (above 0.12). Both 6-phosphogluconate and NADP+ provided good protection against thermal inactivation, with 6-phosphogluconate also providing considerable protection against loss of activity caused by p-chloromercuribenzoate and iodoacetamide. Initial velocity studies indicated the enzyme mechanism was sequential. NADPH was a competitive inhibitor with respect to NADP+, and the Ki values for this inhibition were dependent on the concentration of 6-phosphogluconate. Product inhibition by NADPH was noncompetitive when 6-phosphogluconate was the variable substrate, whereas inhibition by the products CO2 and ribulose 5-phosphogluconate and NADP+ were varied. In totality these data suggest that binding of substrates to the enzyme is random. CO2 and ribulose 5-phosphate are released from the enzyme in random order with NADPH as the last product released.
D001921:Brain / Q000201:enzymology*; D002412:Cations; D002729:Chloromercuribenzoates / Q000494:pharmacology; D005942:Gluconates / Q000378:metabolism; D006801:Humans; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D009994:Osmolar Concentration; D010734:Phosphogluconate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D020245:p-Chloromercuribenzoic Acid
D002412:Cations; D002729:Chloromercuribenzoates; D005942:Gluconates; D009249:NADP; D020245:p-Chloromercuribenzoic Acid; D010734:Phosphogluconate Dehydrogenase; C008884:6-phosphogluconic acid; D007460:Iodoacetamide