An Atomistic Understanding of Allosteric Inhibition of Glutamate Racemase: a Dampening of Native Activation Dynamics

Abstract

Glutamate racemases (GR) are members of the family of bacterial enzymes known as cofactor‐independent racemases and epimerases and catalyze the stereoinversion of glutamate. D‐amino acids are universally important for the proper construction of viable bacterial cell walls, and thus have been repeatedly validated as attractive targets for novel antimicrobial drug design. Significant aspects of the mechanism of this challenging stereoinversion remain unknown. The current study employs a combination of MD and QM/MM computational approaches to show that the GR from H. pylori must proceed via a pre‐activation step, which is dependent on the enzyme's flexibility. This mechanism is starkly different from previously proposed mechanisms. These findings have immediate pharmaceutical relevance, as the H. pylori GR enzyme is a very attractive allosteric drug target. The results presented in this study offer a distinctly novel understanding of how AstraZeneca's lead series of inhibitors cripple the H. pylori GR's native motions, via prevention of this critical chemical pre‐activation step. Our experimental studies, using SPR, fluorescence and NMR WaterLOGSY, show that H. pylori GR is not inhibited by the uncompetitive mechanism originally put forward by Lundqvist et al.. The current study supports a deep connection between native enzyme motions and chemical reactivity, which has strong relevance to the field of allosteric drug discovery.

Racemases and epimerases operating through a 1,1-proton transfer mechanism: reactivity, mechanism and inhibition

“…Exceptions to this strategy are seen with methylmalonyl-CoA epimerase 90 and mandelate racemase, 91,92 where the carboxylate group is ligated to the active site Co 2+ or Mg 2+ ion which acts as a Lewis acid and diminishes the pK a of the C a -H. 21 Typically the carboxylate group is also held within a hydrogen-bonding network with active-site residues. 5 Some racemase/epimerase substrates also contain further destabilising groups, such as ammonium groups (amino-acid racemases/epimerases), 18,20 amide carbonyl groups (N-succinylamino acid racemases, dipeptide epimerases and other enolase family enzymes 18,49 ) and OH (mandelate racemase, 18,91 various sugar epimerases 18 ).…”

“…20,94,97,98 In some enzymes (e.g. glutamate racemase 21 ), this conformational change triggers a change in the conformation of the deprotonating activesite base as part of the pre-activation step which results in protonation of the substrate carboxylate group. It has also been suggested that conformational changes by 'capping domains', which result in the closed form of the racemase, activate the enzyme for catalysis, are important, e.g.…”

“…200 The inhibitor-binding site is remote from the active site. 21,200 A second cryptic inhibitor-binding site was subsequently identified in the B. anthracis enzyme by virtual screening, which led to the identification of pyridine-2,6-dicarboxylate (dipicolinate) 64 as an inhibitor (Fig. 12), with K i = 1.9 mM.…”

“…The enzymes which perform these changes in stereochemical configuration are known as racemases and epimerases, which have been shown to have a pivotal position in metabolism, and thus have gained significant interest as drug targets for diseases such as bacterial infections, 14,[18][19][20][21][22][23][24][25] Chagas disease, [26][27][28] cancer, 6,7,18,29 Alzheimer's disease and other dementias, 1,2,[30][31][32] formation of cataracts 1,33 and diabetic retinopathy; 34 racemase levels are also a marker of ischaemic stroke. 35 Inhibition of diaminopimelate epimerase activity also potentiates cephem antibiotic activity by compromising the integrity of the bacterial cell wall.…”

“…Enzymes which use metal ions as Lewis acids (enolase family enzymes) or are cofactor-independent are of particular interest, since they are able to perform the apparently simple 1,1-proton transfer using active site amino-acid residues and thus are model systems for understanding enzymatic reactions in general. Several of these enzymes are also important as drug targets, 6,7,[20][21][22][23] potential drug targets, 84 or are used in biotechnological applications. 48,49,51 This review will consider racemases/ epimerases utilising deprotonation and deprotonation mechanisms, their reactivity and the strategies used to inhibit them.…”