Dihydroorotase from Escherichia coli. Substitution of Co(II) for the active site Zn(II) - PubMed

Affiliations

• PMID: 1671037

Free article

Dihydroorotase from Escherichia coli. Substitution of Co(II) for the active site Zn(II)

D C Brown et al. J Biol Chem. 1991.

Free article

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.

Similar articles

• Dihydroorotase from Escherichia coli. Sulfhydryl group-metal ion interactions.

  Washabaugh MW, Collins KD. Washabaugh MW, et al. J Biol Chem. 1986 May 5;261(13):5920-9. J Biol Chem. 1986. PMID: 2871020

• Divalent metal derivatives of the hamster dihydroorotase domain.

  Huang DT, Thomas MA, Christopherson RI. Huang DT, et al. Biochemistry. 1999 Aug 3;38(31):9964-70. doi: 10.1021/bi990859x. Biochemistry. 1999. PMID: 10433703

• Molecular structure of dihydroorotase: a paradigm for catalysis through the use of a binuclear metal center.

  Thoden JB, Phillips GN Jr, Neal TM, Raushel FM, Holden HM. Thoden JB, et al. Biochemistry. 2001 Jun 19;40(24):6989-97. doi: 10.1021/bi010682i. Biochemistry. 2001. PMID: 11401542

• Enzyme elements involved in the interconversion of L-carbamylaspartate and L-dihydroorotate by dihydroorotase from Clostridium oroticum.

  Pettigrew DW, Mehta BJ, Bidigare RR, Choudhury RR, Scheffler JE, Sander EG. Pettigrew DW, et al. Arch Biochem Biophys. 1985 Dec;243(2):447-53. doi: 10.1016/0003-9861(85)90521-1. Arch Biochem Biophys. 1985. PMID: 2867744

• The mononuclear metal center of type-I dihydroorotase from Aquifex aeolicus.

  Edwards BF, Fernando R, Martin PD, Grimley E, Cordes M, Vaishnav A, Brunzelle JS, Evans HG, Evans DR. Edwards BF, et al. BMC Biochem. 2013 Dec 9;14:36. doi: 10.1186/1471-2091-14-36. BMC Biochem. 2013. PMID: 24314009 Free PMC article.

Cited by

• Thermoanaerobacter brockii alcohol dehydrogenase: characterization of the active site metal and its ligand amino acids.

  Bogin O, Peretz M, Burstein Y. Bogin O, et al. Protein Sci. 1997 Feb;6(2):450-8. doi: 10.1002/pro.5560060223. Protein Sci. 1997. PMID: 9041649 Free PMC article.

• AtzC is a new member of the amidohydrolase protein superfamily and is homologous to other atrazine-metabolizing enzymes.

  Sadowsky MJ, Tong Z, de Souza M, Wackett LP. Sadowsky MJ, et al. J Bacteriol. 1998 Jan;180(1):152-8. doi: 10.1128/JB.180.1.152-158.1998. J Bacteriol. 1998. PMID: 9422605 Free PMC article.

• Purine and pyrimidine nucleotide synthesis and metabolism.

  Moffatt BA, Ashihara H. Moffatt BA, et al. Arabidopsis Book. 2002;1:e0018. doi: 10.1199/tab.0018. Epub 2002 Apr 4. Arabidopsis Book. 2002. PMID: 22303196 Free PMC article. No abstract available.

• Role of adenine deaminase in purine salvage and nitrogen metabolism and characterization of the ade gene in Bacillus subtilis.

  Nygaard P, Duckert P, Saxild HH. Nygaard P, et al. J Bacteriol. 1996 Feb;178(3):846-53. doi: 10.1128/jb.178.3.846-853.1996. J Bacteriol. 1996. PMID: 8550522 Free PMC article.

• A computational knowledge-base elucidates the response of Staphylococcus aureus to different media types.

  Seif Y, Monk JM, Mih N, Tsunemoto H, Poudel S, Zuniga C, Broddrick J, Zengler K, Palsson BO. Seif Y, et al. PLoS Comput Biol. 2019 Jan 9;15(1):e1006644. doi: 10.1371/journal.pcbi.1006644. eCollection 2019 Jan. PLoS Comput Biol. 2019. PMID: 30625152 Free PMC article.

Publication types

MeSH terms

Substances

LinkOut - more resources

• Full Text Sources

  • Elsevier Science

• Molecular Biology Databases

  • BioCyc