Catalysis and binding in L-ribulose-5-phosphate 4-epimerase: A comparison with L-fuculose-1-phosphate aldolase

L-Ribulose-5-phosphate (L-Ru5P) 4-epimerase and L-fuculose-1-phosphate (L-F uc1P) aldolase are evolutionarily related enzymes that display 26% sequence identity and a very high degree of structural similarity. They both employ a divalent cation in the formation and stabilization of an enolate during catalysis, and both are able to deprotonate the C-4 hydroxyl group of a pho sphoketose substrate. Despite these many similarities, subtle distinctions must be present which allow the enzymes to catalyze two seemingly different reactions and to accommodate substrates differing greatly in the position of the phosphate (C-5 vs C-1). Asp76 of the epimerase corresponds to the ke y catalytic acid/base residue Glu73 of the aldolase. The D76N mutant of the epimerase retained considerable activity, indicating it is not a key catal ytic residue in this enzyme. In addition, the D76E mutant did not show enha nced levels of background aldolase activity. Mutations of residues in the p utative phosphate-binding pocket of the epimerase (N28A and K42M) showed dr amatically higher values of K-M for L-Ru5P. This indicates that both enzyme s utilize the same phosphate recognition pocket, and since the phosphates a re positioned at opposite ends of the respective substrates, the two enzyme s must bind their substrates in a reversed or "flipped" orientation. The ep imerase mutant D120N displays a 3000-fold decrease in the value of k(cat), suggesting that Asp 120' provides a key catalytic acid/base residue in this enzyme. Analysis of the D120N mutant by X-ray crystallography shows that i ts structure is indistinguishable from that of the wild-type enzyme and tha t the decrease in activity was not simply due to a structural perturbation of the active site. Previous work [Lee, L.V., Poyner, R.R., Vu, M.V., and C leland, W.W. (2000) Biochemistry 39, 4821-4830] has indicated that Tyr229' likely provides the other catalytic acid/base residue. Both of these residu es are supplied by an adjacent subunit. Modeling Of L-Ru5P into the active site of the epimerase structure suggests that Tyr229' is responsible for de protonating L-Ru5P and Asp 120' is responsible for deprotonating its epimer , D-Xu5P.