pH profiles of 3-chymotrypsin-like protease (3CLpro) from SARS-CoV-2 elucidate its catalytic mechanism and a histidine residue critical for activity.

3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pH-dependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 pKa values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pKa values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity.

Concerted motion of structure and active site charge is required for tyrosine aminotransferase activity in Leishmania parasite.

Leishmania donovani tyrosine aminotransferase (LdTAT) is an essential enzyme that catalyzes the first step of amino acid catabolism. To understand LdTAT activity at different pH, molecular dynamics simulations were performed and trajectory and T-pad analysis pad were conducted. Fluorescence spectroscopy of LdTAT at various pH was measured to understand structural stability. UV studies on PLP were performed to determine the binding of the enzyme to cofactor PLP at different pH. The MD simulations showed that the structure of LdTAT was stable and no structural denaturation was observed at pH 2, 7 and 12. LdTAT exhibited the highest activity at pH -8 and fluorescent spectroscopy also corroborated by exhibiting the highest intensity at pH -8. Moreover, no structural denaturation was observed during the pH gradient. UV studies concluded that the aldimine bond forms only around neutral pH and redshift was observed on enzyme binding. From our observation, we hypothesize that the activity of LdTAT is a close interplay between the structure and charges of K286 and PLP. This study may provide significant insight into understanding parasitic enzymes like LdTAT during the life-cycle of Leishmania parasite. Knowledge of such enzyme mechanisms can pave the way for the design and delivery of enzyme-specific inhibitors.

Scintigraphy in laryngopharyngeal and gastroesophageal reflux disease: a definitive diagnostic test?

AIM: To investigate the utility of scintigraphic studies in predicting response to laparoscopic fundoplication (LF) for chronic laryngopharyngeal reflux symptoms. METHODS: Patients with upper aero-digestive symptoms that remained undiagnosed after a period of 2 mo were studied with conventional pH and manometric studies. Patients mainly complained of cough, sore throat, dysphonia and globus. These patients were imaged after ingestion of 99m-technetium diethylene triamine pentaacetic acid. Studies were quantified with time activity curves over the pharynx, upper and lower oesophagus and background. Late studies of the lungs were obtained for aspiration. Patients underwent LF with post-operative review at 3 mo after surgery. RESULTS: Thirty four patients (20 F, 14 M) with an average age of 57 years and average duration of symptoms of 4.8 years were studied. Twenty four hour pH and manometry studies were abnormal in all patients. On scintigraphy, 27/34 patients demonstrated pharyngeal contamination and a rising or flat pharyngeal curve. Lung aspiration was evident in 50% of patients. There was evidence of pulmonary aspiration in 17 of 34 patients in the delayed study (50%). Pharyngeal contamination was found in 27 patients. All patients with aspiration showed pharyngeal contamination. In the 17 patients with aspiration, graphical time activity curve showed rising activity in the pharynx in 9 patients and a flat curve in 8 patients. In those 17 patients without pulmonary aspiration, 29% (5 patients) had either a rising or flat pharyngeal graph. A rising or flat curve predicted aspiration with a positive predictive value of 77% and a negative predictive value of 100%. Over 90% of patients reported a satisfactory symptomatic response to LF with an acceptable side-effect profile. CONCLUSION: Scintigraphic reflux studies offer a good screening tool for pharyngeal contamination and aspiration in patients with gastroesophageal reflux disease.

Mechanistic studies on the flavin-dependent N6-lysine monooxygenase MbsG reveal an unusual control for catalysis.

The mechanism of Mycobacterium smegmatis G (MbsG), a flavin-dependent l-lysine monooxygenase, was investigated under steady-state and rapid reaction conditions using primary and solvent kinetic isotope effects, substrate analogs, pH and solvent viscosity effects as mechanistic probes. The results suggest that l-lysine binds before NAD(P)H, which leads to a decrease in the rate constant for flavin reduction. l-lysine binding has no effect on the rate of flavin oxidation, which occurs in a one-step process without the observation of a C4a-hydroperoxyflavin intermediate. Similar effects were determined with several substrate analogs. Flavin oxidation is pH independent while the kcat/Km and kred/KD pH profiles for NAD(P)H exhibit single pKa values of ∼6.0, with increasing activity as the pH decreases. At lower pH, the enzyme becomes more uncoupled, producing more hydrogen peroxide and superoxide. Hydride transfer is partially rate-limiting at neutral pH and becomes more rate-limiting at low pH. An inverse solvent viscosity effect on kcat/Km for NAD(P)H was observed at neutral pH whereas a normal solvent viscosity effect was observed at lower pH. Together, the results indicate a unique mechanism where a rate-limiting and pH-sensitive conformational change occurs in the reductive half-reaction, which affects the efficiency of lysine hydroxylation.