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Identification of the C2‐ 1 H histidine NMR resonances in chloramphenicol acetyltransferase by a 13 C‐ 1 H heteronuclear multiple quantum coherence method
Author(s) -
Derrick J.P.,
Lian L.-Y.,
Roberts G.C.K.,
Shaw W.V.
Publication year - 1991
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(91)80219-s
Subject(s) - histidine , heteronuclear molecule , chemistry , imidazole , chloramphenicol acetyltransferase , titration , heteronuclear single quantum coherence spectroscopy , proton , stereochemistry , ligand (biochemistry) , crystallography , nuclear magnetic resonance spectroscopy , enzyme , physics , biochemistry , gene expression , promoter , quantum mechanics , gene , receptor
Chloramphenicol acetyltransferase (CAT) was used to assess the feasibility of study of specific proton resonances in an enzyme of overall molecular mass 75000. [ ring 2‐ 13 C]Histidine was selectively incorporated into the type III chloramphenicol acetyltransferase (CAT III ) using a histidine auxotroph of E. coli . Heteronuclear multiple and single quantum experiments were used to select the C2 protons in the histidyl imidazole ring. One‐ and two‐dimensional spectra revealed six signals out of a total of seven histidine residues in CAT III . pH titration, chemical modification and ligand binding were used to demonstrate that the signal from H195, the histidine at the active site, is not among those observed. Nevertheless, this work demonstrates that selective isotopic enrichment and multiple quantum coherence techniques can be used to distinguish proton resonances in a protein of high molecular mass.