The first activation study of the β-carbonic anhydrases from the pathogenic bacteriaBrucella suisandFrancisella tularensiswith amines and amino acids

Abstract

The activation of the β-class carbonic anhydrases (CAs, EC 4.2.1.1) from the bacteria Brucella suis and Francisella tularensis with amine and amino acids was investigated. BsuCA 1 was sensitive to activation with amino acids and amines, whereas FtuCA was not. The most effective BsuCA 1 activators were L-adrenaline and D-Tyr (K A s of 0.70–0.95 µM). L-His, L-/D-Phe, L-/D-DOPA, L-Trp, L-Tyr, 4-amino-L-Phe, dopamine, 2-pyridyl-methylamine, D-Glu and L-Gln showed activation constants in the range of 0.70–3.21 µM. FtuCA was sensitive to activation with L-Glu (K A of 9.13 µM). Most of the investigated compounds showed a weak activating effect against FtuCA (K A s of 30.5–78.3 µM). Many of the investigated amino acid and amines are present in high concentrations in many tissues in vertebrates, and their role in the pathogenicity of the two bacteria is poorly understood. Our study may bring insights in processes connected with invasion and pathogenic effects of intracellular bacteria.

Coumarin carbonic anhydrase inhibitors from natural sources

“…Overall, the very interesting moieties present in the NP coumarins make them a source of inspiration for medicinal chemists and pharmacologists in the search of new drugs with a safer profile and specific action in a variety of disorders, starting from the infective ones 79 and ending with tumours 80 and inflammatory diseases 81 . For the moment, only the human α-CAs were investigated for their inhibition profile with coumarins, but such enzymes are also present in other organisms, such as pathogenic bacteria, protozoans, and fungi 82–85 . Investigation of coumarin derivatives (including NPCs) against these enzymes may thus lead to interesting developments in the fight of infections produced by such pathogens.…”

Activation studies of the β-carbonic anhydrases from Escherichia coli with amino acids and amines

“…An Sx.18Mv-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used to assay the catalytic activity of various CA isozymes for CO 2 hydration reaction 30 . Phenol red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.5, for α-CAs) 31–34 or TRIS (pH 8.3, for β-CAs) 35–38 as buffers, 0.1 M NaClO 4 (for maintaining constant ionic strength), following the CA-catalyzed CO 2 hydration reaction for a period of 10 s at 25 °C. The CO 2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants.…”

“…Working at substrate concentrations considerably lower than K M ([ S ] ≪ K M ), and considering that [ A ] f can be represented in the form of the total concentration of the enzyme ([ E ] t ) and activator ([ A ] t ), the obtained competitive steady-state equation for determining the activation constant is given by Equation (5) : where v 0 represents the initial velocity of the enzyme-catalyzed reaction in the absence of activator 35–39 . This type of approach to measuring enzyme-ligand interactions is in excellent agreement with recent results from native mass spectrometry measurements 40 .…”

Effect of amino acids and amines on the activity of the recombinant ι-carbonic anhydrase from the Gram-negative bacterium Burkholderia territorii

“…A Sx.18Mv-R Applied Photophysics (Oxford, UK) stopped-flow instrument was used to assay the CA-catalysed CO 2 hydration activity 58 . Phenol red (at a concentration of 0.2 mM) was used as an indicator, working at the absorbance maximum of 557 nm, with 10 mM HEPES, pH 7.5 (for a-CAs and BteCAi) 59,60 or 10 mM TRIS, pH 8.3 (for b-CAs) [61][62][63][64] as buffers, containing 0.1 M NaClO 4 (for maintaining constant ionic strength), following the CA-catalysed CO 2 hydration reaction for a period of 10-100 s at 25 C. The CO 2 concentrations ranged from 1.7 to 17 mM to determine the kinetic parameters and activation constants. For each activator, at least six traces of the initial 5-10% of the reaction were used to determine the initial velocity.…”

“…where v 0 represents the initial velocity of the enzyme-catalysed reaction in the absence of activator [61][62][63][64] . This type of approach to measuring enzyme-ligand interactions is in excellent agreement with recent results from native mass spectrometry measurements 65 .…”