Dihydroorotase from Escherichia coli. Sulfhydryl group-metal ion interactions.
We have obtained 53 mg of 99% pure dihydroorotase from 10.9 g of frozen Escherichia coli pyrC plasmid-containing E. coli cells using a 4-step 16-fold purification procedure, a yield of 60%. We characterize the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (a dimer of subunit molecular weight 38,300 +/- 2,900), high performance liquid chromatography gel sieving, amino acid analysis, amino terminus determination (blocked), and specific activity. The isolated enzyme contains 1 tightly bound essential zinc atom/subunit, and readily but loosely binds 2 additional Zn(II) or Co(II) ions/subunit which modulate catalytic activity; treatment of crude extracts with weak chelators suggests that the enzyme contains 3 zinc atoms/subunit in vivo. Two of the 6 thiol groups/subunit react rapidly with 5,5'-dithiobis(2-nitrobenzoate) when 1 Zn/subunit enzyme is used, but slowly when 3 Zn/subunit enzyme is used. The 2 weakly bound Zn(II) ions/subunit protect against the reversible air oxidation which lowers the specific activity of the enzyme and renders it unreactive with 5,5'-dithiobis(2-nitrobenzoate). The dilution activation observed in the presence of substrate, the dilution inactivation observed in the absence of substrate, and the transient activation by the metal chelator oxalate are interpreted as evidence for an unstable, hyperactive monomer.
Washabaugh
M W
MW
Collins
K D
KD
D000581:Amidohydrolases / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D003035:Cobalt / Q000032:analysis*; D004080:Dihydroorotase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010957:Plasmids; D011485:Protein Binding; D013438:Sulfhydryl Compounds / Q000032:analysis; D015032:Zinc / Q000032:analysis*
D000596:Amino Acids; D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D003035:Cobalt; D004228:Dithionitrobenzoic Acid; D000581:Amidohydrolases; D004080:Dihydroorotase; D015032:Zinc