The mechanism of 2-keto-3-deoxy-6-phosphogluconic aldolase. II. Studies of the partial reactions with substrate analogues
Journal of Biological Chemistry
The specificity of 2-keto-3-deoxy-6-phosphogluconic (KDP-gluconate) aldolase for azomethine formation was studied by incubating a-carbonyl compounds and borohydride with the aldolase and determining the amount of inactivation sustained. Both pyruvate and a-ketobutyrate produced complete inactivation, whereas monohydroxyacetone, a-ketoisovalerate, a-ketoglutarate, 5-keto-4-deoxyglucarate, 2-keto-4-hydroxyglutarate, and 2-keto-3-deoxygluconate were partially inhibitory. Hydroxypyruvate and
... pyruvate and 2-ketogluconate were not inhibitory; thus, it was concluded that the only major restriction upon azomethine formation was against the presence of a hydroxyl group at carbon 3. Formation of an azomethine with a-ketobutyrate prevented subsequent azomethine formation with pyruvate, and a-ketobutyrate under some conditions competed with pyruvate for azomethine formation. Thus, it was concluded that the same lysine residue is involved in azomethine formation for both keto acids. The curves of the effect of concentration of pyruvate and a-ketobutyrate show about a 40-fold lower affinity for a-ketobutyrate. The aldolase catalyzed the exchange of only 1 proton from water into a-ketobutyrate. The initial rate of exchange was at least 37-fold slower with a-ketobutyrate than with pyruvate. 2-Keto-4-hydroxyglutarate was cleaved at 0.1% of the rate with KDP-gluconate, whereas 2-keto-3-deoxygluconate was cleaved at a definite, but much slower, rate. Oxalacetate was decarboxylated at about 0.5% of the rate with KDPgluconate. These data are interpreted as demonstrating a dissociation of azomethine formation, cleavage, and perhaps proton exchange phases of the over-all reaction catalyzed by KDPgluconate aldolase.