Role of Metal Ions in the Reaction Catalyzed by l-Ribulose-5-phosphate 4-Epimerase

Abstract

H97N, H95N, and Y229F mutants of L-ribulose-5-phosphate 4-epimerase had 10, 1, and 0.1%, respectively, of the activity of the wild-type (WT) enzyme when activated by Zn(2+), the physiological activator. Co(2+) and Mn(2+) replaced Zn(2+) in Y229F and WT enzymes, although less effectively with the His mutants, while Mg(2+) was a poorly bound, weak activator. None of the other eight tyrosines mutated to phenylalanine caused a major loss of activity. The near-UV CD spectra of all enzymes were nearly identical in the absence of metal ions and substrate, and addition of substrate without metal ion showed no effect. When both substrate and Zn(2+) were present, however, the positive band at 266 nm increased while the negative one at 290 nm decreased in ellipticity. The changes for the WT and Y229F enzymes were greater than for the two His mutants. With Co(2+) as the metal ion, the CD and absorption spectra in the visible region were different, showing little ellipticity in the absence of substrate and a weak absorption band at 508 nm. With substrate present, however, an intense absorption band at 555 nm (epsilon = 150-175) with a negative molar ellipticity approaching 2000 deg cm(2) dmol(-1) appears with WT and Y229F enzymes. With the His mutants, the changes induced by substrate were smaller, with negative ellipticity only half as great. The WT, Y229F, H95N, and H97N enzymes all catalyze a slow aldol condensation of dihydroxyacetone and glycolaldehyde phosphate with an initial k(cat) of 1.6 x 10(-3) s(-1). The initial rate slowed most rapidly with WT and H97N enzymes, which have the highest affinity for the ketopentose phosphates formed in the condensation. The EPR spectrum of enzyme with Mn(2+) exhibited a drastic decrease upon substrate addition, and by using H(2)(17)O, it was determined that there were three waters in the coordination sphere of Mn(2+) in the absence of substrate. These data suggest that (1) the substrate coordinates to the enzyme-bound metal ion, (2) His95 and His97 are likely metal ion ligands, and (3) Tyr229 is not a metal ion ligand, but may play another role in catalysis, possibly as an acid-base catalyst.

Enzymatic Characterization of 5-Methylthioribulose-1-phosphate Dehydratase of the Methionine Salvage Pathway inBacillus subtilis

“…FucA, RibE, and RhuA are members of the divalent metal iondependent class II aldolase family. [13][14][15][16][17][18] These proteins, including MtnB, possess three histidine residues binding a metal ion, a glutamate or aspartate residue subtracting a proton from their substrates, and residues binding the phosphate group from the substrate (Fig. 8).…”

“…[13][14][15][16][17][18] There has been no report on the dehydration activity of enzymes belonging to this family. In this, the enzymatic relationship of MtnB to this family of enzymes is discussed.…”

Utilization of l -Ascorbate by Escherichia coli K-12: Assignments of Functions to Products of the yjf-sga and yia-sgb Operons

“…Both SgaE and SgbE catalyzed the epimerization of L-ribulose 5-phosphate to D-xylulose 5-phosphate, using the established coupled enzyme assay for AraD (transketolase, triose phosphate isomerase, and ␣-glycerolphosphate dehydrogenase [9][10][11]). The kinetic constants for the SgaE-and SgbE-catalyzed reactions, presented in Table 2, are comparable to those reported for AraD (9-11) (values not shown), so we conclude that SgaE and SgbE are L-ribulose 5-phosphate 4-epimerases.…”

TM0416, a Hyperthermophilic Promiscuous Nonphosphorylated Sugar Isomerase, Catalyzes Various C 5 and C 6 Epimerization Reactions

“…The catalytic mechanism and biochemical properties of phosphorylated sugar epimerases have been reviewed extensively (22,23). Phosphorylated sugar epimerases can be divided into several groups, according to their mechanism of catalysis, i.e., (i) proton abstraction/addition (24,25), (ii) formation of a transient keto intermediate (26,27), (iii) carbon-carbon bond cleavage (28,29), (iv) mutarotation (ring opening) (30), and (v) nucleotide elimination and readdition (31). However, little is known about the nonphosphorylated L-ribulose 3-epimerase (R3E).…”

“…1D). Several sugar epimerases that are involved in the pentose phosphate pathway require divalent metal ions for epimerization via a cis-enediolate-stabilized intermediate (29). Therefore, to investigate the effects of divalent metal ions on TM0416 activity, we purified a metal-depleted enzyme by using EDTA treatment followed by dialysis to yield the apo form of TM0416 (with an estimated 0.015 Ϯ 0.001 equivalents of Mn 2ϩ per subunit), which was verified using high-resolution inductively coupled plasma mass spectrometry (ICP-MS).…”