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Human Brain 6‐Phosphogluconate Dehydrogenase: Purification and Kinetic Properties
Author(s) -
Weisz Karen S.,
Schofield Philip J.,
Edwards Michael R.
Publication year - 1985
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1985.tb05443.x
Subject(s) - dehydrogenase , enzyme , non competitive inhibition , chemistry , product inhibition , biochemistry , ribulose , substrate (aquarium) , glucose 6 phosphate dehydrogenase , enzyme assay , iodoacetamide , stereochemistry , biology , rubisco , ecology , cysteine
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 V max and a decrease in K m 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 K i 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 CO 2 and ribulose 5‐phosphate was noncompetitive when both 6‐phosphogluconate and NADP + were varied. In totality these data suggest that binding of substrates to the enzyme is random. CO 2 and ribulose 5‐phosphate are released from the enzyme in random order with NADPH as the last product released.