Cobalt(III) carboxypeptidase A

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

doi:10.1021/ja00856a021

Cited by 24 publications

(12 citation statements)

References 8 publications

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“…Oxidation of [(CPD)Co(II)] with Hydrogen Peroxide. The preparation of [(CPD)60Co(III)] was attempted by treating [(CPD)60Co(Il)] with hydrogen peroxide according to the procedure of Kang et al (1975). The effect of this procedure on the peptidase and esterase activities of the enzyme toward Cbz-Gly-L-Phe and Bz-Gly-L-OPhe, respectively, as a function of time is shown in Figure 1.…”

Section: Resultsmentioning

confidence: 99%

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Enzymatically inactive, exchange-inert cobalt(III)-carboxypeptidase A: role of inner sphere coordination in peptide and ester catalysis

Wart¹,

Vallée²

1978

Biochemistry

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Catalytically inactive, exchange-inert Co(III)-carboxypeptidase A has been prepared by reaction of Co(II)-carboxypeptidase A with the active-site-directed oxidizing agent m-chloroperbenzoic acid. Co(III)-carboxypeptidase A, isolated by affinity gel filtration chromatography, has the same amino acid composition and molecular weight as the starting material and contains 0.95 g-atom/mol of cobalt and 0.01 g-atom/mol of zinc. Its electron paramagnetic resonance, circular dichroic, magnetic circular dichroic, and visible absorption spectra are consistent with those of octahedral Co(III) model complexes. Co(III)-caboxypeptidase A is essentially devoid of catalytic activity toward both peptide and ester substrates of the native enzyme, and stopped-flow fluorescence studies with dansylated substrates show that it binds peptides, but not esters. Furthermore, the protein does not react with either type of substrate to yield a single turnover. The implications of these findings to the mechanism of action of carboxypeptidase A are discussed in the light of the "metal-carbonyl" and "metal-hydroxide" hypotheses. Since Co(III)-carboxypeptidase A does not bind esters, inner-sphere coordination to the metal appears to be necessary for ester binding. All attempts to prepare Co(III)-carboxypeptidase A by treatment of Co(II)-carboxypeptidase A with hydrogen peroxide according to previously published procedures (Kang, E.P., Storm, C.B., & Carson, F.W. (1975) J. Am. Chem. Soc. 97, 6723) have been unsuccessful, and the present results do not confirm earlier reports that Co(III)-carboxypeptidase A exhibits esterase activity or that its activity is dependent on the method of preparation of the precursor Co(II)-carboxypeptidase A (Jones, M.M., Hunt, J.B., Storm, C.B., Evans, P.S., Carson, F.W. & Pauli, W.J. (1977) Biochem. Biophys. Res. Commun. 75, 253). These findings call for a reexamination of mechanistic conclusions based on the assumption that Co(III)-carboxypeptidase A is an active esterase.

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Section: Resultsmentioning

confidence: 99%

“…of the unmodified controls. The enzyme, 1.08 X 10~4 M in 1 M NaCl, 0.1 M Tris-HCl, pH 7.5, was treated with 1 mM hydrogen peroxide at 22 °C, according to the procedure of Kang et al (1975). (See also Figures 2 and 4 in Piras & Vallee, 1966a.…”

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confidence: 99%

Enzymatically inactive, exchange-inert cobalt(III)-carboxypeptidase A: role of inner sphere coordination in peptide and ester catalysis

Wart¹,

Vallée²

1978

Biochemistry

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“…One modification is the substitution of other metal ions for Zn2+ (6); the effects on catalysis are not parallel for ester and peptide substrates. A particularly striking example is the report (7) that replacement of Zn2+ by Co3+, which is inert to substitution in its first coordination sphere, leads to an enzyme that has lost all peptidase activity but still retains esterase activity. Other studies (8) (12).…”

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confidence: 99%

Unified picture of mechanisms of catalysis by carboxypeptidase A.

Breslow¹,

Wernick²

1977

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

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ABSTRACT. It was centrifuged, washed with water, and dissolved in 10 mM N-ethylmorpholine/acetic acid buffer at pH 7.5, which was 2.0 M in LiCl, then diluted with an equal volume of 170 mM buffer and centrifuged. Activity was assayed with benzoylglycylphenylalanine (12). Oxygen-18-enriched substrates were prepared by hydrolysis of the corresponding methyl esters with H2180 and sodium methoxide (N-acetyl-L-phenylalanine 25% 180 in one carboxyl oxygen, N-acetylglycine 16% enriched, and N-benzoylglycine 75% enriched) or by hydrolysis of the acid chloride in H2180 (p-chloro-trans-cinnamic acid 6.8% unlabeled, 92.7% singly labeled, 0.46% doubly labeled).Oxygen-18 exchange studies were performed in pH 7.50 170 mM N-ethylmorpholine/acetic acid buffer (,u = 0.1). Identical samples containing substrate, enzyme, and cosubstrate, if any, were made up. One was immediately freeze-dried while the other was incubated for the desired time, assayed for residual enzyme activity, then freeze-dried (the buffer is volatile). Isotopic abundance was determined with a chemical ionization mass spectrometer under a multiple ion detection program averaged over 50 scans. In the case of acetylglycine, the products were esterified with ethereal diazomethane prior to analysis by gas chromatography-chemical ionization mass spectrometry to avoid interfering peaks from other reaction components.Unlabeled substrates were prepared in standard ways, and the novel ones were fully characterized: N-acetyl-L-phenylalanine methyl ester, mp 86-88°; p-chloro-trans-cinnamoyl-L-phenylalanine, mp 211-212°. O-[3H]Acetyl-L-mandelic acid was prepared as described for the untritiated compound (13)

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“…A binding constant of 2 X M was determined by Hunt & Ginsburg for Co(I1) binding to the "tight" metal ion site but Co(I1) binding to the second binding site was not determined. Villafranca et al (1978) determined binding constants for Co(I1) to the n, and n2 metal ion sites with adenylylated glutamine synthetase and the values were K , = 1.4 X M. Co(II1)-glutamine synthetase was prepared by oxidation of Co(I1)-glutamine synthetase similar to that adopted for Co(II1)-carboxy peptidase A (Kang et al, 1975). Apoglutamine synthetase (0.18-0.23 m M subunit concentration) was equilibrated with a 1:l stoichiometric amount of CoC1, that contained a suitable amount of 57C0 for radioassay ( N 1.4 X lo5 cpm).…”

Section: Methodsmentioning

confidence: 99%