Galactose-1-phosphate uridylyltransferase from Escherichia coli, a zinc and iron metalloenzyme.
Galactose-1-P uridylyltransferase purified from Escherichia coli cells grown in enriched medium contains approximately 1.2 mol of tightly bound zinc/mol of subunits as well as variable amounts of iron, up to 0.7 mol/mol of subunits, and no detectable Ca, Cd, Cu, Mo, Ni, Co, Mn, As, Pb, or Se. The chelators, 1,10-phenanthroline, 8-hydroxyquinoline, 8-hydroxyquinoline sulfonate, and 2,2'-bipyridyl remove metal ions from the enzyme and allow the importance of zinc and iron to be evaluated. Dialysis of this enzyme against 2 mM 1,10-phenanthroline, 8-hydroxyquinoline sulfonate, and 2,2'-bipyridyl at millimolar concentrations slowly removes both zinc and iron from the enzyme (t1/2 = 4 days at 24 degrees C) with concomitant loss of enzymatic activity. In chelation experiments utilizing 1,10-phenanthroline, residual enzymatic activity was found to be proportional to the zinc content, to the iron content, and to the sum of zinc and iron. UDP-glucose (0.35 mM) protects the enzyme against loss of metal ions and activity in the presence of 1,10-phenanthroline, whereas glucose-1-P at 70 mM (400 x Km) fails to protect. The enzyme purified from cells grown on a minimal medium containing inorganic salts and glucose supplemented with either ZnSO4 or FeSO4 shows approximately the same level of enzymatic activity as the enzyme from cells grown on enriched medium. These experiments showed that enzymatic activity is supported by either iron or zinc associated with two sites in the enzyme. Enzyme depleted of metal ions by chelators can be partially reactivated by addition of ZnSO4.(ABSTRACT TRUNCATED AT 250 WORDS)
Ruzicka
F J
FJ
Institute for Enzyme Research, Graduate School, University of Wisconsin-Madison 53705, USA.
Wedekind
J E
JE
Kim
J
J
Rayment
I
I
Frey
P A
PA
D000596:Amino Acids / Q000032:analysis; D002104:Cadmium / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003300:Copper / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D007501:Iron / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008345:Manganese / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008670:Metals / Q000032:analysis; D010957:Plasmids; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D015032:Zinc / Q000032:analysis* / Q000378:metabolism
D000596:Amino Acids; D046911:Macromolecular Substances; D008667:Metalloproteins; D008670:Metals; D002104:Cadmium; D008345:Manganese; D003300:Copper; D007501:Iron; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase; D015032:Zinc