Role of metal ions in the reaction catalyzed by L-ribulose-5-phosphate 4-epimerase

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 wh en activated by Zn2+, the physiological activator. Co2+ and Mn2+ replaced Z n2+ in Y229F and WT enzymes, although less effectively with the His mutants , while Mg2+ was a poorly bound, weak activator. None of the other eight ty rosines 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 e ffect. When both substrate and Zn2+ were present, however, the positive ban d at 266 nm increased while the negative one at 290 nm decreased in ellipti city. The changes for the WT and Y229F enzymes were greater than for the tw o His mutants. With Co2+ aS the metal ion, the CD and absorption spectra in the visible region were different, showing little ellipticity in the absen ce of substrate and a weak absorption band at 508 nm. With substrate presen t, however, an intense absorption band at 555 nm (epsilon = 150-175) with a negative molar ellipticity approaching 2000 deg cm(2) dmol(-1) appears wit h WT and Y229F enzymes. With the His mutants, the changes induced by substr ate were smaller, with negative ellipticity only half as great. The WT: Y22 9F, H95N, and H97N enzymes all catalyze a slow aldol condensation of dihydr oxyacetone 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, w hich have the highest affinity for the ketopentose phosphates formed in the condensation. The EPR spectrum of enzyme with Mn2+ exhibited a drastic dec rease upon substrate addition, and by using (H2O)-O-17, it was determined t hat there were three waters in the coordination sphere of Mn2+ in the absen ce of substrate. These data suggest that (1) the substrate coordinates to t he 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 ill c atalysis, possibly as an acid-base catalyst.