Inhibition of Urease, a Ni‐Enzyme: The Reactivity of a Key Thiol With Mono‐ and Di‐Substituted Catechols Elucidated by Kinetic, Structural, and Theoretical Studies

Abstract

The inhibition of urease from Sporosarcina pasteurii (SPU) and Canavalia ensiformis (jack bean, JBU) by a class of six aromatic poly‐hydroxylated molecules, namely mono‐ and dimethyl‐substituted catechols, was investigated on the basis of the inhibitory efficiency of the catechol scaffold. The aim was to probe the key step of a mechanism proposed for the inhibition of SPU by catechol, namely the sulfanyl radical attack on the aromatic ring, as well as to obtain critical information on the effect of substituents of the catechol aromatic ring on the inhibition efficacy of its derivatives. The crystal structures of all six SPU‐inhibitors complexes, determined at high resolution, as well as kinetic data obtained on JBU and theoretical studies of the reaction mechanism using quantum mechanical calculations, revealed the occurrence of an irreversible inactivation of urease by means of a radical‐based autocatalytic multistep mechanism, and indicate that, among all tested catechols, the mono‐substituted 3‐methyl‐catechol is the most efficient inhibitor for urease.

Inhibition of Urease by Hydroquinones: A Structural and Kinetic Study

“…In this equation, the k 1 and k 2 constants are functions of the single kinetic constants that regulate each step of the inactivation mechanism as well as the concentration of the inhibitor (Scheme 1). In order to quantitatively compare the inhibition strengths of HQ, MHQ, and t BHQ, the values of the k 1 ⋅ k 2 products determined for the data collected at a single and shared inhibitor concentration (1.6 mM) are reported in Table 5, as already done for catechol and its mono‐ and di‐methyl derivatives [26,27] . The resulting analysis showed that the inactivation proficiency of the hydroquinone derivatives follows the order HQ≃MHQ> t BHQ.…”

“…Kinetic measurements : Pre‐incubation experiments were conducted at 25 °C as previously described [24,26,27] . In brief, a solution of 50 μg mL −1 active JBU dissolved in 20 mM HEPES buffer, pH 7.5, was 100‐fold diluted in Cresol Red (CR) buffer, consisting of 30 mg L −1 CR dissolved in 2 mM HEPES buffer (pH 7.5), 2 mM EDTA.…”

“…While such a comparison of k 1 •k 2 products was possible within the HQ, MHQ, and tBHQ triad, a similar quantitative assessment of the relative urease inhibition efficiency between catechols and hydroquinones cannot be carried out because of the much lower inhibition efficiency of the latter, which prevents the possibility to perform the experiments in the same range of concentrations for the two classes of compounds. However, a qualitative comparison was carried out by evaluating the concentration required to achieve a 50 % decrease of Chemistry-A European Journal urease activity in a range of 10 2 -10 3 seconds: such an inhibition level is achieved at 20 μM by catechols, [26][27] and 1.6 mM by hydroquinones, reflecting an overall approximately two orders of magnitude greater inhibition efficiency of the former with respect to the latter. The overall higher reactivity of catechols can be rationalized on the basis of the different reduction potentials for the oxidized/semireduced/reduced forms of the two classes of compounds: [40] assuming that the same radical-based autocatalytic multistep urease inactivation mechanism is operational in both cases (Scheme 1), the starting reduced forms must undergo a one-electron oxidation step in order for the active species, namely their corresponding semiquinone moieties, to be generated.…”

Organosulfur Compounds as Soil Urease Inhibitors and Their Effect on Kinetics of Urea Hydrolysis

“…It can either directly bind to the nickel ions in the urease active site or modify specific cysteine residue located on the mobile flap covering the dinickel center of the enzyme (Svane et al 2020;Mazzei et al 2020). The flap is essential for urease activity as it controls substrate access to the active site (Mazzei et al 2016).…”

A small-molecular inhibitor against Proteus mirabilis urease to treat catheter-associated urinary tract infections

“…Furthermore, the proposed 2-MA inhibitor contains a thiol moiety, which is known to display active inhibition of the urease enzyme, as described elsewhere 23 , 24 . Urease inhibitors have been extensively examined by previous researchers as described by 25 , 26 . However, many focus on their use in agriculture and few test their use on medicinally relevant bacterial ureases such as those from P. mirabilis .…”