Abstract
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.
Highlights
From the Departmentof Biological Chemistry, the Uniueiv i t y of Maryland Medical School, Baltimore, Maryland21201
Homodimer of subunit molecular weight 38,729) con- membered pyrimidine ring(involving the P-carboxyl groupof taining only the 1 activesite Zn(I1) ion persubunit with the sulfhydryl reagent N-(ethyl)-maleimide (NEM)blocks the two externalZn(I1)sites per subunit and dramatically lessens the precipitation caused by high concentrations of Zn(I1); stabilizes the enzyme partially against air oxidation and dilution inactivation; makes the active siteZn(1I)easier to remove; and lowers K, and increases kcat
Treatment ofNEMblocked dihydroorotase ((NEM)dihydroorotase)with the chelator 2,6-pyridinedicarboxylicacid at pH 5.0 in the absence of oxygen and trace metal ions removes the active site Zn(l1)with a half-life of 15 min, allowing the production of milligram amountsof moderately
Summary
From the Departmentof Biological Chemistry, the Uniueiv i t y of Maryland Medical School, Baltimore, Maryland21201. Treatment ofNEMblocked dihydroorotase ((NEM)dihydroorotase)with the chelator 2,6-pyridinedicarboxylicacid at pH 5.0 in the absence of oxygen and trace metal ions removes the active site Zn(l1)with a half-life of 15 min, allowing the production of milligram amountsof moderately. The E. coli pyrC gene has been sequenced (Backstrom et al, 1986; Wilson et al, 1987).The enzyme is a homodimer of subunit molecular weight38,729’ with a tightly bound catalyticZn(I1) ionat the active site (Washabaugh and Collins, 1984); it has 2 weakly bound structural Zn(I1). Alway of the pyrimidines, has a number of interestingand unusual properties It is onoef the few enzymes used biosyntheticallytosynthesizeanamidebondwithout adirectly coupled energy source such as ATP hydrolysis to drive the reaction; the cyclization of N-(carbamyl)-L-aspartate,the reaction catalyzed, occurs readily because it is favored entropically relativeto themore commonintermolecular amide bond formation. In the absence of the enzyme the amide cyclization reaction product is the 5-membered hydantoin
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