Characterization of Bovine Carboxypeptidase A (Allan)

Ph, Pétra; Ma, Hermodson; Ka, Walsh; Neurath, Hans

doi:10.1021/bi00798a600

Cited by 22 publications

(15 citation statements)

References 15 publications

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“…4. The oxidation of Co(I1)-CPA reported here was carried out under very mild conditions, the molar ratio of hydrogen peroxide to protein being about 10 and the temperature 22O.…”

Section: Discussionmentioning

confidence: 97%

Cobalt(III) carboxypeptidase A

Kang¹,

Storm²,

Carson³

1975

J. Am. Chem. Soc.

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60) Since V, is oriented perpendicular to the Fe-N4 plane in 20, the same orientation might be expected in 19. The observation of preferential ori-(1967).(62) M. M.'Maltempo. Electronic spectra are not informative in this respect due to the spin-forbidden nature of ligand field transitions and the intense charge-transfer and ligand-based absorptions throughout the uv-visible region. Spectral Abstract: Cobalt(II1) carboxypeptidase A has been prepared by the oxidation of cobalt(I1) carboxypeptidase A with hydrogen peroxide at pH 7.5. The oxidized enzyme shows negligible peptidase activity after dialysis against metal-free buffer or 1, IO-phenanthroline while its esterase activity toward hippuryl-L-0-phenyllactate and trans-p-nitrocinnamoyl-L-&phenyllactate is comparable to that of cobalt(I1) carboxypeptidase A. Carbobenzyloxyglycyl-L-phenylalanine, toward which the cobalt(I1) enzyme is active, competitively inhibits the activity of the cobalt(II1) protein toward hippuryl-D,L-p-phenyllactate. The metal in the oxidized enzyme is no longer removable by dialysis against metal-free buffer, but may be reduced back to cobalt(I1) and exchanged by dialysis against excess cobalt(I1). Cobalt(iI1) carboxypeptidase A has a visible absorption maximum at 503 nm (t 500). The pH dependence of ester hydrolysis by cobalt(II1) carboxypeptidase A shows that catalysis requires the basic form of a group on the enzyme with pKa 6.3-6.5 and the acidic form of a group with pK, 9.1-9.5. These results have a bearing on the mechanism of action of carboxypeptidase A. In particular, they imply that the group of higher pK, is not a water molecule bound to the metal and that ligation of substrates within the first coordination sphere of the metal does not occur during ester hydrolysis catalyzed by cobalt(II1) carboxypeptidase A.

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“…4. The oxidation of Co(I1)-CPA reported here was carried out under very mild conditions, the molar ratio of hydrogen peroxide to protein being about 10 and the temperature 22O.…”

Section: Discussionmentioning

confidence: 97%

Cobalt(III) carboxypeptidase A

Kang¹,

Storm²,

Carson³

1975

J. Am. Chem. Soc.

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“…The a and j# forms of carboxypeptidase are much more soluble than the y enzyme (10). Hence, different conditions, critical for labeling them with a single arsanilazotyrosine-248 residue per molecule (9) had to be used for each form and crystal type of the enzyme.…”

Section: Methodsmentioning

confidence: 99%

Conformations of Arsanilazotyrosine-248 Carboxypeptidase A _α,β,γ . Comparison of Crystals and Solution

Johansen

Vallée

1973

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

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The spectra of the a, ,, and -y forms of zinc monoarsanilazotyrosine-248 carboxypeptidase A are indistinguishable. At pH 8.2 their crystals are yellow, while their solutions are red, Xmax 510 nm. Absorption and circular dichroism-pH titrations of the modified zinc and apoenzymes demonstrate that the absorption band at 510 nm is due to a complex between arsanilazotyrosine-248 and the active-site zinc atom. Two pKapp values, 7.7 and 9.5, characterize the formation and dissociation of this arsanilazotyrosine-248-Zn complex. On titrations of the apoenzyme, the absorption band at 510 nm is completely absent at all pH values. Instead, there is a single pKapp, 9.4, due to the ionization of the azophenol, Xxs 485 nm. Substitution of other metals for zinc results in analogous intramolecular coordination complexes with absorption maxima and circular dichroism extrema characteristic of the particular metal. Similar data and conclusions have been derived from studies of heterocyclic azophenol metal complexes.The present studies demonstrate that the conformation of the crystals of all generally available a, A, and My forms of the arsanilazoenzyme differs from that of their solutions. The spectra of the modified x-ray crystals, however, differ from those of all other carboxypeptidase forms and crystal habits studied. The internal consistency of their data, their interpretation, and the conclusions of Lipscomb and coworkers [Proc. Nat. Acad. Sci. USA (1972) 69, 2850-2854] are examined. Dissimilar chemical modification or conformation is thought to underlie these differences.The arsanilazotyrosine-248 -zinc complex is a sensitive, dynamic probe of environmental conditions. Its response to changes in pH and physical state of the enzyme suggest different orientation of the arsanilazotyrosine-248 side chain in solution from that in the crystal. This finding calls for reexamination of the basis of the substrate-induced conformation change which has been thought to be critical to the mechanism, postulated on the basis of the x-ray structure analysis performed at pH 7.5.The integration of functional data obtained in solution with the structure derived from crystals remains one of the important problems in discerning the mechanism of action of an enzyme. We have searched for means that in solution could simultaneously gauge activity and the structural dynamics of the active center under a wide range of environmental conditions. Several chromophoric probes have been found particularly helpful in this regard (1,2).In relating the specific structure of carboxypeptidase Aa EC 3.4.2.1 to the catalytic mechanism of carboxypeptidase in general, the location of Tyr-248 with respect to the active-site Zn atom has been thought critical (3, 4). When specifically coupled with diazotized arsanilic acid, solutions of carboxypeptidase Ay exhibit an absorption spectrum with a maximum at 510 nm (red) thought to be indicative of an intramolecular coordination complex between mono-arsanilazotyrosine-248 and Znt. This complex can be dissociated a...

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“…The possible reaction of WRK with carboxyl groups in E. coli or S. cerevisiae PEPCKs was not addressed in this work. In this respect, it is interesting to point out that Petra and Neurath (1971) successfully used WRK and [14C]-methoxamine to label a carboxyl group at the active site of carboxypeptidase A. Similarly, Jennings and Smith (1992) used [3H]NaBH4 as a secondary label of the WRK-derivatized carboxyl groups of human red blood cell band 3 protein, identifying Glu-681 as the reactive residue.…”

Section: Characterization Of Wrk-modified Pepcksmentioning

confidence: 99%

“…Later, WRK was used to identify carboxylate side chains in trypsin (Bod- 467 laender et al, 1969) and in carboxypeptidase A (Petra and Neurath, 1971). Since then WRK has been used as a probe for reactive carboxylate groups in many enzymes (Lundbland and Noyes, 1984;Vangrysperre et al, 1989;Maralihalli and Bhagwat, 1993;Keresztessy et al, 1994;Chauthaiwale and Rao, 1994;Komissarov et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

Woodward's reagent K reacts with histidine and cysteine residues inEscherichia coli andSaccharomyces cerevisiae phosphoenolpyruvate carboxykinases

et al. 1996

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The reaction of Woordward's reagent K (WRK) with model amino acids and proteins has been analyzed. Our results indicate that WRK forms 340-nm-absorbing adducts with sulfhydryl- and imidazol-containing compounds, but not with carboxylic acid derivatives, in agreement with Liamas et al. [(1986), J. Am. Chem. Soc. 108, 5543-5548], but not with Sinha and Brewer [(1985), Anal. Biochem. 151, 327-333]. The chemical modification of Escherichia coli and Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases with WRK leads to an increase in the absorption at 340 nm, and we have demonstrated its reaction with His and Cys residues in these proteins. These results caution against claims of glutamic or aspartic acid modification by WRK based on the absorption at 340 nm of protein- WRK adducts.

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Characterization of Bovine Carboxypeptidase A (Allan)

Cited by 22 publications

References 15 publications

Cobalt(III) carboxypeptidase A

Cobalt(III) carboxypeptidase A

Conformations of Arsanilazotyrosine-248 Carboxypeptidase A _α,β,γ . Comparison of Crystals and Solution

Woodward's reagent K reacts with histidine and cysteine residues inEscherichia coli andSaccharomyces cerevisiae phosphoenolpyruvate carboxykinases

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Characterization of Bovine Carboxypeptidase A (Allan)

Cited by 22 publications

References 15 publications

Cobalt(III) carboxypeptidase A

Cobalt(III) carboxypeptidase A

Conformations of Arsanilazotyrosine-248 Carboxypeptidase A α,β,γ . Comparison of Crystals and Solution

Woodward's reagent K reacts with histidine and cysteine residues inEscherichia coli andSaccharomyces cerevisiae phosphoenolpyruvate carboxykinases

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Conformations of Arsanilazotyrosine-248 Carboxypeptidase A _α,β,γ . Comparison of Crystals and Solution