Chemistry and photochemistry attending the inactivation of Escherichia coli β-hydroxydecanoyl thiol ester dehydrase by an acetylenic diazoketone

Article abstract of DOI:10.1021/ja00091a001

β-Hydroxydecanoyl thiol ester dehydrase from Escherichia coli, an enzyme that catalyzes both dehydration and allylic rearrangement reactions, has been shown previously to undergo mechanism-based inactivation by the acetylenic substrate analog 1-diazo-4-undecyn-2-one (DUO). Details of the chemistry and photochemistry of DUO are now presented. Analysis of DUO-inactivated dehydrase by ¹⁵N NMR spectrosocpy indicates that DUO quantitatively alkylates histidine-70. Long-wavelength photoirradiation leads to spectrophotometrically observable changes in the DUO-dehydrase adduct. The structural changes were characterized by a combination of methods. Samples of protein that had been inactivated with [1-¹³C]-, [2-¹³C]-, and [3-¹³C] DUO were analyzed by ¹³C NMR spectroscopy, both prior and subsequent to photoirradiation. By comparisons of the chemical shifts of the enriched atoms of the inactivator moiety with those of model compounds, it was confirmed that the α-diazoketone moiety is retained in DUO-inactivated dehydrase and that photoirradiation leads to Wolff rearrangement of the α-diazoketone moiety followed by attack of a nucleophile on the resulting ketone. Proteolytic degradation of photoirradiated, DUO-inactivated dehydrase gave peptides that were analyzed by Edman sequencing and by mass spectrometry. The results are consistent with a single protein modification (at H70), and attack on the ketone by water, leading to a carboxylic acid. Samples of intact native, DUO-inactivated, and photoirradiated, DUO-inactivated dehydrase were analyzed by electrospray ionization mass spectrometry, the results of which clearly support the hypothesis that the ketone suffers attack by water. In light of recent results from X-ray crystallography, it is proposed that the water that attacks the ketone is one of two bound water molecules with specific roles in the binding and/or catalytic turnover of substrate. A Michael addition-elimination mechanism is presented to explain the unexpected hydrolytic lability of the DUO-dehydrase adduct.