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J. Biol. Chem., Vol. 262, Issue 19, 9016-9020, 07, 1987
JV Miller, DA Estell and RA Lazarus
2,5-Diketo-D-gluconate reductase, a novel enzyme that catalyzes the
stereospecific NADPH-dependent reduction of 2,5-diketo-D-gluconate to 2-
keto-L-gulonate, has been purified to homogeneity by sequential anion
exchange, Cibacron blue F3GA affinity, and gel permeation chromatography
from Corynebacterium sp. ATCC 31090. Molecular weight of the native form,
determined by gel permeation chromatography, is 35,000 +/- 2,000. The
subunit molecular weight, determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis is 34,000; thus, the enzyme is
active as a monomer. A pI value of 4.4 is measured for the enzyme. Amino-
and carboxyl-terminal sequences are consistent with that predicted by the
DNA sequence of the reductase gene. At 25 degrees C, pH 6.4, the turnover
number is 500 min-1, and the apparent Km values for 2,5-diketo-D-gluconate
and NADPH are 26 mM and 10 microM, respectively. The enzyme is specific for
NADPH, but the sugar binding site will also accept 5-keto-D-fructose and
dihydroxyacetone as substrates. The enzyme is active over a broad pH range
(pH 5-8) for the reduction of 2,5-diketo-D-gluconate; a sharp optimum at pH
9.2 is observed for the oxidation of 2-keto-L-gulonate. A Keq value of 5.6
X 10(-13) M indicates that reduction of substrate by NADPH is highly
preferred. An activation energy of 12.3 kcal mol-1 is measured. Enzyme
turnover is slow relative to dehydration of the gem-diol at C-5 of the
substrate.
Purification and characterization of 2,5-diketo-D-gluconate reductase from Corynebacterium sp
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