Dihydroorotase from Escherichia coli. Substitution of Co(II) for the active site Zn(II).
Treatment of Escherichia coli dihydroorotase (a homodimer of subunit molecular weight 38,729) containing only the 1 active site Zn(II) ion per subunit with the sulfhydryl reagent N-(ethyl)-maleimide (NEM) blocks the two external Zn(II) sites per subunit and dramatically lessens the precipitation caused by high concentrations of Zn(II); stabilizes the enzyme partially against air oxidation and dilution inactivation; makes the active site Zn(II) easier to remove; and lowers Km and increases kcat. Treatment of NEM-blocked dihydroorotase ((NEM)dihydroorotase) with the chelator 2,6-pyridinedicarboxylic acid at pH 5.0 in the absence of oxygen and trace metal ions removes the active site Zn(II) with a half-life of 15 min, allowing the production of milligram amounts of moderately stable apo-(NEM)dihydroorotase in about 80% yield. Treatment of apo-(NEM)dihydroorotase with Co(II) at pH 7.0 produces (NEM)dihydroorotase completely substituted at the active site with Co(II) in 100% yield: analysis gives 0.95-1.1 g atoms of Co(II) per active site and 0.03-0.05 g atoms of Zn(II) per active site. This Co(II)-(NEM)dihydroorotase is hyperactive at pH 8. The electronic absorption spectrum of Co(II)-(NEM)dihydroorotase at pH 6.5 implicates an active site thiol group as a ligand to the metal ion. The spectrum is inconsistent with tetrahedral coordination of the active site metal ion and is most consistent with a pentacoordinate structure.
Brown
D C
DC
Department of Biological Chemistry, University of Maryland Medical School, Baltimore 21201.
Collins
K D
KD
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000378:metabolism; D001665:Binding Sites; D002413:Cations, Divalent; D003035:Cobalt / Q000378:metabolism*; D004080:Dihydroorotase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D013057:Spectrum Analysis; D015032:Zinc / Q000378:metabolism*
D001059:Apoproteins; D002413:Cations, Divalent; D003035:Cobalt; D008345:Manganese; D004080:Dihydroorotase; D015032:Zinc; D005033:Ethylmaleimide