Ca^II Binding Regulates and Dominates the Reactivity of a Transition‐Metal‐Ion‐Dependent Diesterase from Mycobacterium tuberculosis

Abstract

The diesterase Rv0805 from Mycobacterium tuberculosis is a dinuclear metallohydrolase that plays an important role in signal transduction by controlling the intracellular levels of cyclic nucleotides. As Rv0805 is essential for mycobacterial growth it is a promising new target for the development of chemotherapeutics to treat tuberculosis. The in vivo metal-ion composition of Rv0805 is subject to debate. Here, we demonstrate that the active site accommodates two divalent transition metal ions with binding affinities ranging from approximately 50 nm for Mn(II) to about 600 nm for Zn(II) . In contrast, the enzyme GpdQ from Enterobacter aerogenes, despite having a coordination sphere identical to that of Rv0805, binds only one metal ion in the absence of substrate, thus demonstrating the significance of the outer sphere to modulate metal-ion binding and enzymatic reactivity. Ca(II) also binds tightly to Rv0805 (Kd ≈40 nm), but kinetic, calorimetric, and spectroscopic data indicate that two Ca(II) ions bind at a site different from the dinuclear transition-metal-ion binding site. Ca(II) acts as an activator of the enzymatic activity but is able to promote the hydrolysis of substrates even in the absence of transition-metal ions, thus providing an effective strategy for the regulation of the enzymatic activity.

Product release is rate-limiting for catalytic processing by the Dengue virus protease

“…pK es1 ) and the catalytic efficiency (i.e. pK e1 )2829303132. The main difference between the unlinked and glycine-linked systems is that for the former the activity at low pH continues to decrease while for the latter it reaches a final value (at pH 7 the respective rates are 0.1 s −1 and 0.4 s −1 ; Fig.…”

The Evolution of New Catalytic Mechanisms for Xenobiotic Hydrolysis in Bacterial Metalloenzymes

“…[39,[42][43][44][45] The significance of outer residues is demonstrated in the differing metal ion specificities and reaction mechanisms observed between the diesterase Rv0805 from Mycobacterium tuberculosis and the enzyme GpdQ from Enterobacter aerogenes despite these having identical first-shell coordination of the respective metal ions. [46] This also demonstrates the intrinsic challenge associated with the generation of biomimetics that match the catalytic properties of their biological representatives. [47] Owing to this apparent optimised structural specificity, it has often been assumed that metalloenzymes are specific for the metal ion they are bound to when purified.…”

Kinetic, mechanistic, structural and spectroscopic investigations of Bimetallic Metallohydrolases

“…This is supported by recently published atomic absorption measurements of Mn 2+ or Co 2+ -reconstituted CpsB and ITC data, which also indicate the presence of only two bound metal ions in the active enzyme (266) . The metal ion affinities, determined by ITC, are similar to those of the well-characterised antibiotic-degrading MBL AIM-1 (72) (see also Chapter 5) and cyclic nucleotide diesterases, (257) but significantly weaker than those recorded for phosphotriesterases. (232) To date the in vivo metal ion composition of CpsB remains obscure, but Mn 2+ appears to be the most likely candidate, based on optimal catalytic performance combined with bioavailability.…”

“…The electron density suggests that the third metal ion (M3) might be weakly bound due to its lower occupancy. Although uncommon, catalytic active sites possessing more than two metal ion have been seen before for some metallohydrolases (231) . In terms of the reaction mechanism it has been proposed that the catalytic cycle in CpsB involves an esterolysis-initiating hydroxide nucleophile that is doubly Lewis-activated by coordination to M1 and M2.…”

“…A solution of 5 mM of the desired metal ion was then titrated into the reaction cell, with a spacing of 60 s between injections to allow the heat in the cell to return to the baseline. The data were analysed using MicroCal Origin 7.0 software using an approach described elsewhere (195,231) . The heat released during each injection was fitted to an equation that was derived from a model including two individual binding sites (195,231) .…”