Assay ofEscherichia coliDihydroorotase with Enantiomeric Substrate: Practical Preparation of Carbamyll-Aspartate and High-Performance Liquid Chromatography Analysis of Catalysis Product

Daniel, Régis; Kokel, B.; Caminade, Eliane; Martel, Annie; Goffic, F. Le

doi:10.1006/abio.1996.0307

Cited by 7 publications

(1 citation statement)

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“…) (10) in the biosynthetic conversion of CA to DHO as well as in the degradative pathway of DHO into CA as reported previously (11). Protein concentrations were determined by the Bio-Rad dye binding method with bovine serum albumin as the standard (12) and referred to concentrations of subunits (i.e.…”

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confidence: 99%

Mass Spectrometric Determination of the Cleavage Sites inEscherichia coli Dihydroorotase Induced by a Cysteine-specific Reagent

Daniel

Caminade

Martel

et al. 1997

Journal of Biological Chemistry

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Escherichia coli dihydroorotase contains six cysteines/subunit, which are potential ligands of structural and catalytic zinc metals at protein sites of the enzyme. Specific thiol reagents modify, in nondenaturing conditions only, two of these cysteines; these two residues are thought to be ligands of structural zinc. We report here on the localization of these two cysteines on the polypeptide chain through their cyanylation by 2-nitro-5-thiocyanobenzoic acid (NTCB) and the analysis by mass spectrometry of the protein adducts. This is the first study of E. coli dihydroorotase by mass spectrometry, allowing the accurate determination of the subunit molecular weight (38,695). Treatment of dihydroorotase by NTCB induced a cleavage N-terminal to the cyanylated cysteines. The resulting fragments visualized on electrophoresis gel have been N-terminal sequenced, and their masses were determined by electrospray-ionizing mass spectrometry. This allowed the identification of cysteines 221 and 265 as the two residues cyanylated by the reagent NTCB. Results from gel filtration of dihydroorotase cyanylated on the two cysteines indicate that these residues are involved in subunit interactions leading to the active dimer. Consistent with literature data, we assume that cysteine 221 and cysteine 265, along with the neighboring cysteines 263 and 268 arranged in cluster, are potential ligands of structural zinc of E. coli dihydroorotase.

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confidence: 99%