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 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.
Lee
L V
LV
Institute for Enzyme Research, Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53705, USA.
Poyner
R R
RR
Vu
M V
MV
Cleland
W W
WW
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis / Q000187:drug effects; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology; D002942:Circular Dichroism; D003035:Cobalt / Q000378:metabolism / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism / Q000494:pharmacology; D008670:Metals / Q000378:metabolism / Q000494:pharmacology*; D008956:Models, Chemical; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012274:Ribulosephosphates / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014867:Water / Q000378:metabolism; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
D002413:Cations, Divalent; D008024:Ligands; D008670:Metals; D012274:Ribulosephosphates; D014867:Water; D003035:Cobalt; C031524:ribulose 5-phosphate; D008345:Manganese; D005634:Fructose-Bisphosphate Aldolase; D002238:Carbohydrate Epimerases; C022554:L-ribulosephosphate 4-epimerase; D008274:Magnesium; D015032:Zinc