MECHANISM-BASED INACTIVATION OF ESCHERICHIA-COLI BETA-HYDROXYDECANOYL THIOL ESTER DEHYDRASE - ASSIGNMENT OF IMIDAZOLE N-15 NMR RESONANCES AND DETERMINATION OF THE STRUCTURE OF THE ALKYLATED HISTIDINE

Nitrogen-15 nuclear magnetic resonance spectroscopy was used to determine the structure of the active-site histidine-70 adduct formed when beta-hydroxydecanoyl thiol ester dehydrase from Escherichia coli reacts with the mechanism-based inactivator S-(3-decynoyl)-N-acetylcysteamine (3-decynoyl-NAC). In order to obtain the amount of labeled enzyme necessary for spectral studies, the fabA gene, which encodes dehydrase, was overexpressed to give dehydrase as 15-20% of soluble protein. To simplify the interpretation of the NMR spectra, the non-active-site histidine residue His-129 was converted to an asparagine residue using site-directed mutagenesis. The specific activity and response of the mutant to 3-decynoyl-NAC are unaltered. It is known that type beta (''pyridine-like'') nitrogens in imidazoles resonate 60-80 ppm downfield of type alpha (''pyrrole-like'') nitrogens. To assign the imidazole nitrogen resonances in dehydrase, wild-type and mutant enzymes were labeled with (NH4Cl)-N-15 or with [N-15delta1]histidine. Analysis of the N-15 NMR spectra allowed the assignment of the resonances of the imidazole nitrogens of His-129 and His-70. For His-70, the spectra show that Ndelta1 resonates upfield of Nepsilon2 in the native enzyme and is therefore a type alpha nitrogen. In the inactivated enzyme the signals are reversed, and Nepsilon2 is a type alpha nitrogen. These results demonstrate that Nepsilon2 of His-70 becomes alkylated upon inactivation of dehydrase with 3-decynoyl-NAC and thus is the probable locus of active-site basicity in the normal reactions catalyzed by dehydrase. In addition, the imidazole nitrogen chemical shifts suggest that Ndelta1 may bc involved in a hydrogen bond in native dehydrase but that Nepsilon2 is not. The mechanistic implications of this are discussed.