The Mechanism of 2-Keto-3-deoxy-6-phosphogluconate Aldolase

Abstract 2-Keto-3-deoxy-6-phosphogluconate aldolase of Pseudomonas putida reacts with about 4 moles of 1-fluoro-2,4-dinitrobenzene per mole of aldolase; this is accompanied by 85 to 90% loss in activity. Both the arylation reaction and the inactivation are prevented by 2-keto-3-deoxy-6-phosphogluconate, 2-keto-3-deoxy-6-phosphogalactonate, and inorganic phosphate. The substrate binding site was then blocked by adding 2-keto-3-deoxy-6-phosphogalactonate and reducing the azomethine linkage to a lysine residue with sodium borohydride. Subsequent dialysis of the treated enzyme to remove noncovalently bound 2-keto-3-deoxy-6-phosphogalactonate, followed by reaction with fluorodinitrobenzene, results in only one of the four originally observed dinitrophenylations. This indicates that 3 molecules of fluorodinitrobenzene react in or close to the active site. Only the lysine e-amino group is dinitrophenylated, and these lysines were shown to be distinct from those forming an azomethine with pyruvate, 2-keto-3-deoxy-6-phosphogalactonate, and, presumably, 2-keto-3-deoxy-6-phosphogluconate. The Km of dinitrophenylated aldolase for 2-keto-3-deoxy-6-phosphogluconate is about 2-fold higher than that of native aldolase, whereas there is a 10-fold decrease in Vmax. Thus, the loss in activity by dinitrophenylation seen at saturating 2-keto-3-deoxy-6-phosphogluconate concentration is attributed to a decrease in catalysis rather than to a decrease in binding of 2-keto-3-deoxy-6-phosphogluconate. The rate of tritium exchange from T2O into pyruvate decreased about 10-fold following dinitrophenylation. Since tritium exchange in native 2-keto-3-deoxy-6-phosphogluconate aldolase is only 2 to 3 times the rate of cleavage, the 10-fold loss of activity may be attributed to either direct or indirect impairment of the C—H bond cleavage part of the reaction mechanism. Discrepancies between the rate and extent of dinitrophenylation and the loss in activity were observed. These are attributed to a conformational change elicited by addition of the first molecule of fluorodinitrobenzene. An altered conformation is also supported by the unexpected change in pI to a more basic region, increased thermal stability, and decreased number of sulfhydryl groups available for titration with dithiodinitrobenzoate. The data do not support the hypothesis that dinitrophenylation impairs the substrate phosphate group binding site but, rather, that it effects changes in conformation which indirectly diminish catalysis.