Cobalt (III) carboxypeptidase A: Preparation and esterase activity

Kang, Eunju; Storm, Carlyle B.; Carson, Frederick W.

doi:10.1016/0006-291x(72)90456-1

Cited by 25 publications

(15 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To the best of the authors' knowledge, this is only the third report that an active enzyme-Co 3+ complex has been formed. Kang and Storm (1972) reported that the Co 3+…”

Section: Discussionmentioning

confidence: 98%

“…Reports of stable cation-protein complexes with Co 3+ (Kang & Storm, 1972;Shinar & Navon, 1974;Anderson & Vallee, 1975;Balakrishnan & Villafranca, 1975;Miziorko et al, 1982) have suggested the utility of Co 3+ as a probe of the PEPCK metal activator site. The spectral properties, preference for nitrogen and oxygen donors, and inertness to substitution reactions render Co 3+ a potentially valuable probe for such a study.…”

Section: Discussionmentioning

confidence: 99%

“…The in situ oxidation of Co 2+ has been used to study the metal binding sites of several proteins. Kang and Storm (1972) first reported the initial preparation and characterization of Co 3+ -carboxypeptidase A via the oxidation of the Co 2+ -enzyme with H 2 O 2 . The Co 3+ in carboxypeptidase A is ligand exchange-inert, and the modified enzyme has low peptidase activity.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Formation and Characterization of an Active Phosphoenolpyruvate Carboxykinase−Cobalt(III) Complex

Hlavaty

Nowak

1997

Biochemistry

View full text Add to dashboard Cite

Avian mitochondrial phosphoenolpyruvate carboxykinase (PEPCK) was incubated with Co2+ and H2O2 to form a stable Co3+-PEPCK complex. PEPCK, similarly incubated with H2O2 and either Mg2+ or Mn2+, resulted in no significant loss in activity over 30 min. PEPCK, incubated with Co2+ and H2O2 at pH 7.4, showed rapid inhibition as observed by a 40% decrease in activity after 5 min. The loss of activity is linear with the incorporation of cobalt into PEPCK, resulting in 15-25% activity for the stoichiometric Co3+-PEPCK complex. The incorporation of and inhibition by Co3+ is protected by PEP and GTP (ITP). Treatment of the Co3+-PEPCK complex with beta-mercaptoethanol results in a loss of cobalt and full recovery of activity. The reduction and reactivation are protected by PEP and GTP (ITP). EPR, PRR, circular dichroism, and fluorescence studies all indicate that Co3+ has been selectively incorporated into the cation site of PEPCK, resulting in a catalytically active enzyme-cation species. The substrates form Michaelis complexes with Co3+-PEPCK, and the catalytic reaction occurs as a second sphere complex as previously suggested [Lee & Nowak (1984) Biochemistry 23, 6506); Duffy & Nowak (1985) Biochemistry 24, 1152]. Proteolytic digestion of the Co3+-PEPCK complex and isolation of the cobalt-containing peptide by reverse phase HPLC were performed to identify the location of the cation binding site. From mass, amino acid composition, and sequence analyses of the isolated cobalt-peptide, the region Thr276-Lys301 is responsible for metal chelation. This very homologous region, located in the central portion of PEPCK, contains two highly conserved aspartic acids, Asp295 and Asp296, that are the only feasible metal binding ligands.

show abstract

“…To the best of the authors' knowledge, this is only the third report that an active enzyme-Co 3+ complex has been formed. Kang and Storm (1972) reported that the Co 3+…”

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Formation and Characterization of an Active Phosphoenolpyruvate Carboxykinase−Cobalt(III) Complex

Hlavaty

Nowak

1997

Biochemistry

View full text Add to dashboard Cite

show abstract

“…However, oxidation transforms Co(II) from the exchange-labile (d7) to the exchange-inert (d6) Co(III) state. Preliminary evidence for carboxypeptidase (5) and carbonic anhydrase (6) indicates that the cobalt in Co(II)-substituted enzymes can be oxidized to Co(III), which is exchange-inert.…”

mentioning

confidence: 99%

Cobalt(III), a probe of metal binding sites of Escherichia coli alkaline phosphatase.

Anderson¹,

Vallée²

1975

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

To facilitate the study of individual metal binding sites of polymeric metalloproteins, conversion of exchange-labile Co(II) in E. coli alkaline phosphatase (EC 3.1.3.1) to exchange-inert Co(III) Alkaline phosphatase [EC 3.1.3.1; orthophosphoric-monoester phosphohydrolase(alkaline optimum)] isolated from Escherichia coli contains 4 g-atoms of zinc per molecular weight 89,000. One pair of zinc atoms is involved primarily in catalysis, while the second pair appears to play a predominantly structural role (1). The zinc atoms can be replaced by Co(II), Ni(II), Cu(II), Cd(II), Mn(JI), Fe(II), and Hg(II) but, by steady-state kinetics, only the cobalt-substituted enzyme is catalytically active (2). While the zinc enzyme is both a phosphohydrolase and a phosphotransferase, the cobalt enzyme displays only hydrolase activity (3). This enzymatically active, cobalt-substituted enzyme also exhibits a very characteristic visible absorption spectrum (1).All of the metal ions that have been demonstrated to bind to alkaline phosphatase are exchange-labile (4). However, oxidation transforms Co(II) from the exchange-labile (d7) to the exchange-inert (d6) Co(III) state. Preliminary evidence for carboxypeptidase (5) and carbonic anhydrase (6) indicates that the cobalt in Co(II)-substituted enzymes can be oxidized to Co(III), which is exchange-inert.Chromophoric metal atoms at the active sites of metalloenzymes, e.g., alkaline phosphatase, exhibit spectral properties distinct from those at structural or regulatory sites (7,8

show abstract

“…More recently, Ka~g and Storm (171) have shown that conversion of cobalt(II) carboxypeptidase A to the corresponding cobalt (III) enzyme by hydrogen peroxide oxidation is accompanied by the complete loss of peptidase activity when assayed with CbzGly-L-Phe. Nevertheless, the esterase activity, as measured by the rate of hydrolysis of O-(N-benzoyl-Gly)-D,L-phenyllactic acid, remains unaffected by this transformation.…”

Section: Bovine Carboxypeptidase Amentioning

confidence: 99%

Mechanisms of zinc ion catalysis in small molecules and enzymes

Dunn

Structure and Bonding

View full text Add to dashboard Cite

Cobalt (III) carboxypeptidase A: Preparation and esterase activity

Cited by 25 publications

References 14 publications

Formation and Characterization of an Active Phosphoenolpyruvate Carboxykinase−Cobalt(III) Complex

Formation and Characterization of an Active Phosphoenolpyruvate Carboxykinase−Cobalt(III) Complex

Cobalt(III), a probe of metal binding sites of Escherichia coli alkaline phosphatase.

Mechanisms of zinc ion catalysis in small molecules and enzymes

Contact Info

Product

Resources

About