Probing the coenzyme and substrate binding events of CDP-D-glucose 4,6-dehydratase: Mechanistic implications

NAD(+)-dependent nucleotidyl diphosphohexose 4,6-dehydratases which transform nucleotidyl diphosphohexoses into the corresponding 4-keto-6-deoxy sugar derivatives are essential to the formation of all 6-deoxyhexoses. Studies of the CDP-D-glucose 4,6-dehydratase (E(od)) from Yersinia had shown that this dimeric protein binds only 1 equiv of NAD(+)/mol of enzyme and, unlike other enzymes of the same,class, displays a unique NAD(+) requirement for full catalytic activity. Analysis of the primary sequence revealed an extended ADP-binding fold (GHTGFKG) which deviates from the common Rossmann consensus (GXGXXG) and thus may have contributed to E(od)'s limited NAD(+) affinity. In particular, the presence of His17 in the beta-turn region and that of Lys21 in a position typically occupied by a small hydrophobic residue may impose electronic or steric perturbations to this essential binding motif. To better understand the correlation between the binding properties and primary sequence, mutants (H17G and K21I) were constructed to provide enzymes containing an ADP binding region which more closely resembles the Rossmann-type fold. Analysis of the cofactor and substrate binding characteristics of the wild-type and mutant enzymes helped define the presence of two binding sites for both CDP-D-glucose and NAD(+) per enzyme molecule. While both mutants displayed enhanced NAD(+) affinity, the H17G mutation resulted in an enzyme with slightly higher k(cat) and a 3-fold increase in catalytic efficiency (k(cat)/K-m). The large anticooperativity found for NAD(+) binding (K-1 = 40.3 +/- 0.4 nM, K-2 = 539.8 +/- 4.8 nM) may explain why the cofactor binding sites of wild-type E(od) are only half-occupied. Further examination also revealed the purified E(od) to contain sequestered NADH and that the affinity of E(od) for NADH (K-1 = 0.21 +/- 0.01 nM, K-2 = 7.46 +/- 0.25 nM) is much higher than that for NAD(+). Thus, it is possible that E(od)'s half-site saturation of NAD(+) per enzyme dimer may also be attributed to a significant portion of the cofactor binding sites being occupied by NADH. Interestingly, the sequestered NADH is released upon binding with CDP-D-glucose, These results implicate a new kinetic mechanism for E(od) catalysis.