Mechanistic analysis of enzymes from pathogenic bacteria

Abstract

The first part of this thesis focuses on the kinetic and chemical mechanism of arylamine N-acetyltransferase from Mycobacterium tuberculosis (TBNAT). Arylamine N-acetyltransferases (NATs) are cytosolic enzymes that catalyze the transfer of the acetyl group from acetyl coenzyme A to the free amino group of arylamines and hydrazines. Previous studies have reported that overexpression of NAT from Mycobacterium smegmatis and Mycobacterium tuberculosis may be responsible for increased resistance to the most powerful antitubercular drug, isoniazid, by acetylating and hence inactivating the prodrug. We report the kinetic characterization of TBNAT, which reveals that substituted anilines are excellent substrates for the enzyme. Conversely, isoniazid is a very poor substrate for TBNAT, thus suggesting that expression of this enzyme does not contribute to isoniazid resistance. Steady-state kinetic analysis of TBNAT reveals that the enzyme catalyzes the reaction via a Bi-Bi ping-pong kinetic mechanism. The pH dependence of the kinetic parameters and the analysis of solvent kinetic isotope effects provide sufficient evidence to propose a chemical mechanism for TBNAT. In the last chapter of Part 1, we suggest future experiments that may validate and exploit TBNAT as a novel drug target for antitubercular therapy.;Iron acquisition, via the biosynthesis and secretion of small molecule iron-chelating siderophores, is crucial for the virulence of most pathogenic bacteria. Inhibition of this pathway, which is unique to bacteria, represents a promising strategy for antibacterial drug development. The second part of this thesis describes kinetic and inhibition studies of dihydroxybenzoate-AMP ligase (EntE) from Escherichia coli, an enzyme essential for the biosynthesis of the siderophore, enterobactin. EntE catalyzes the ATP-dependent transfer of 2, 3-dihydroxybenzoic acid (DHB) onto the phosphopantetheinylated cofactor that is attached to the aryl carrier protein domain (ArCP) of EntB to yield the covalently arylated EntB-ArCP, a product that is essential for the final assembly of enterobactin. Steady-state analysis reveals that EntE proceeds via a Bi-Uni-Uni-Bi ping-pong kinetic mechanism and additionally, we show the order of substrate binding and product release. Interestingly, we discovered that EntE, when in the absence of phosphopantetheinylated EntB-ArCP, catalyzes the formation of P¹, P³-diadenosine-5'-tetraphosphate (Ap4A), a modulator of metabolism during cellular stress. We propose that the EntE-catalyzed formation of Ap4A may provide additional aid to iron-depleted cells such that, while enterobactin is scavenging ferric iron, Ap4A may act to moderate cellular activities until sustainable iron concentrations are restored.;Steady-state analysis of two hydrolytically-stable adenylate analogues, Sal-AMS and DHB-AMS, reveals that they act as slow-onset tight-binding inhibitors that bind EntE with low nanomolar and picomolar affinities. Direct binding experiments confirm low picomolar dissociation constants for both analogues to EntE. The tight-binding of Sal-AMS and DHB-AMS to EntE suggests that these compounds may be starting scaffolds for further elaboration of rationally-designed antibiotics targeted to this enzyme. Our data suggest that inhibition of EntE, in addition to abolishing enterobactin biosynthesis, may also hinder cellular stress-response mechanisms, as we have shown that EntE also has the capacity to catalytically produce Ap4A. In the final chapter of Part II, we propose additional experiments that may be completed to test this hypothesis and validate the in vivo biosynthesis of Ap4A by EntE.