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

TitleCatalysis and binding in L-ribulose-5-phosphate 4-epimerase: A comparison with L-fuculose-1-phosphate aldolase
Publication TypeJournal Article
Year of Publication2001
AuthorsSamuel, J, Luo, Y, Morgan, PM, Strynadka, NCJ, Tanner, ME
Date PublishedDec
Type of ArticleArticle
ISBN Number0006-2960

L-Ribulose-5-phosphate (L-Ru5P) 4-epimerase and L-fuculose-1-phosphate (L-Fuc1P) 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 phosphoketose 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 key catalytic acid/base residue Glu73 of the aldolase. The D76N mutant of the epimerase retained considerable activity, indicating it is not a key catalytic residue in this enzyme. In addition, the D76E mutant did not show enhanced levels of background aldolase activity. Mutations of residues in the putative phosphate-binding pocket of the epimerase (N28A and K42M) showed dramatically higher values of K-M for L-Ru5P. This indicates that both enzymes utilize the same phosphate recognition pocket, and since the phosphates are positioned at opposite ends of the respective substrates, the two enzymes must bind their substrates in a reversed or "flipped" orientation. The epimerase 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 its structure is indistinguishable from that of the wild-type enzyme and that 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 Cleland, W.W. (2000) Biochemistry 39, 4821-4830] has indicated that Tyr229’ likely provides the other catalytic acid/base residue. Both of these residues are supplied by an adjacent subunit. Modeling Of L-Ru5P into the active site of the epimerase structure suggests that Tyr229’ is responsible for deprotonating L-Ru5P and Asp 120’ is responsible for deprotonating its epimer, D-Xu5P.

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