Inactivation of beta-hydroxydecanoyl thiolester dehydrase by a photoreactive mechanism-based inactivator

Barry Steven Henderson, Purdue University

Abstract

$\beta$-Hydroxydecanoyl thiolester dehydrase, the pivotal enzyme in the biosynthesis of unsaturated fatty acids in Escherichia coli, interconverts thiolesters of (R)-3-hydroxydecanoic acid, E-2-decenoic acid, and Z-3-decenoic acid. Consideration of the p$K\sb{\rm a}$ of the conjugate acid of His-70 (the sole active site base involved in deprotonation steps at C-2, C-3, and C-4 of the substrates) and those of dehydrase's substrates has led to speculation that an acidic active site residue may assist the deprotonation of substrate by His-70 through hydrogen bonding to or protonation of the substrate carbonyl oxygen. Evidence for such a group was sought through the use of 1-diazo-4-undecyn-2-one (DUO), a potential active site cross-linking reagent. DUO irreversibly inactivates dehydrase, and protection by substrate indicates that this process is an active site directed process. Moreover, one molecule of (2-$\sp{14}$C) DUO is incorporated per active site. A primary deuterium isotope effect of 5.2 is observed with (3,3-$\sp2$H$\sb2$) DUO, indicating that the inactivation involves a rate limiting proton abstraction at C-3. Using a combination of $\sp{15}$N and $\sp{13}$C NMR, the initial inactivation was shown to result in the tethering of the $\alpha$-diazomethyl ketone moiety to His-70, most likely via a vinyl imidazole linkage. Photoirradiation of the enzyme-bound $\alpha$-diazomethyl ketone results in a Wolff rearrangement to produce a ketene, which is attacked by a nearby nucleophile. Peptide mapping studies and electrospray mass spectrometry indicate that this nucleophile is water.

Degree

Ph.D.

Advisors

Schwab, Purdue University.

Subject Area

Biochemistry

COinS