Dehydroquinate synthase: the use of substrate analogues to probe the early steps of the catalyzed reaction.

The early steps of the proposed mechanistic pathway for dehydroquinate synthase have been probed with a series of substrate analogues. These analogues, 3-9, are structurally prohibited from undergoing the beta-elimination of inorganic phosphate that represents the committed step in the conversion of the substrate 3-deoxy-D-arabino-heptulosonate 7-phosphate (1) to dehydroquinate (2). In agreement with previous observations, the analogues that possess shortened side chains (3,5, and 6) bind more tightly to the enzyme than those (4 and 7-9) that are more nearly isosteric with the substrate. Two hitherto unrecognized factors that influence binding have been identified: (i) carbacylic analogues bind 25-100 times more tightly than the corresponding oxacyclic materials (indeed, the carbacyclic phosphonate 5 has a Ki value of 8 x 10(-10)M) and (ii) the side chain appears to be bound in a gauche conformation similar to the most stable conformation of the cis-vinylhomophosphonate 8. These trends in binding can be rationalized by considering the behavior of the analogues in the first two chemical steps of the mechanism: NAD+-mediated oxidation at C-5 and enolization at C-6 (the first part of the E1cB elimination of inorganic phosphate). Direct spectrophotometric determination of the equilibrium level of enzyme-bound NADH indicates that the carbacyclic analogues are more readily oxidized than the oxacyclic compounds, and this predictable difference in redox behavior is reflected in the observed differences in binding. The gauche conformation of the C-7 side chain appears to be required for proton abstraction from C-6, since only those analogues that can adopt this conformation undergo enzyme-catalyzed exchange of the C-6 proton with the solvent. This conformation positions one of the peripheral oxygens of the phosphate (or phosphonate) group close to the C-6 proton. Taken together with other data, these results suggest that the enzyme exploits this substrate base in the enolization, which occurs through an intramolecular proton transfer. The loss of Pi then completes the beta-elimination.