IDENTIFICATION OF THE C2-H-1 HISTIDINE NMR RESONANCES IN CHLORAMPHENICOL ACETYLTRANSFERASE BY A C-13-H-1 HETERONUCLEAR MULTIPLE QUANTUM COHERENCE METHOD

Chloramphenicol acetyltransferase (CAT) was used to assess the feasibility of study of specific proton resonances in an enzyme of overall molecular mass 75 000. [ring 2-C-13]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.