Kinetic Analysis Misinterpretations Due to the Occurrence of Enzyme Inhibition by Reaction Product: Comparison between Initial Velocities and Reaction Time Course Methodologies

The Michaelis–Menten equation (MME) has been extensively used in biochemical reactions, but it is not appropriate when the reaction product inhibits the enzyme. Under these circumstances, each determined initial velocity, v0, is one experimental point that actually belongs to a different MME because enzymatic product inhibition occurs as the reaction starts. Furthermore, the inhibition effect is not constant, since the concentration of the product inhibitor rises as time increases. To unveil the hidden enzyme inhibition and to simultaneously demonstrate the superiority of an integrated Michaelis–Menten equation (IMME), the same range of data points, assuming product inhibition and the presence of a second different inhibitor, was used for kinetic analysis with both methodologies. This study highlights the superiority of the IMME methodology for when the enzyme is inhibited by the reaction product, giving a more coherent inhibition model and more accurate kinetic constants than the classical MME methodology.

25-Hydroxycholesterol-Conjugated EK1 Peptide with Potent and Broad-Spectrum Inhibitory Activity against SARS-CoV-2, Its Variants of Concern, and Other Human Coronaviruses

The COVID-19 pandemic caused by SARS-CoV-2 infection poses a serious threat to global public health and the economy. The enzymatic product of cholesterol 25-hydroxylase (CH25H), 25-Hydroxycholesterol (25-HC), was reported to have potent anti-SARS-CoV-2 activity. Here, we found that the combination of 25-HC with EK1 peptide, a pan-coronavirus (CoV) fusion inhibitor, showed a synergistic antiviral activity. We then used the method of 25-HC modification to design and synthesize a series of 25-HC-modified peptides and found that a 25-HC-modified EK1 peptide (EK1P4HC) was highly effective against infections caused by SARS-CoV-2, its variants of concern (VOCs), and other human CoVs, such as HCoV-OC43 and HCoV-229E. EK1P4HC could protect newborn mice from lethal HCoV-OC43 infection, suggesting that conjugation of 25-HC with a peptide-based viral inhibitor was a feasible and universal strategy to improve its antiviral activity.

Identification of a TeO32− reductase/mycothione reductase from Rhodococcus erythropolis PR4

ABSTRACT A Rhodococcus erythropolis bacterium that tolerates normally lethal concentrations of Fe(II), Cu(II), AsO32−, SeO32−, TeO32−, Cd(II) and Zn(II) was identified from an environmental isolate. In characterizing the molecular basis for metal tolerance, a mycothione reductase (Mtr) with remarkable selectivity for TeO32− reduction over SeO32− was identified. In equimolar concentrations of TeO32− and SeO32−, the enzymatic product contains a 7-fold excess of Te. This selectivity is remarkable because the standard reduction potential of SeO32− is 0.20 V more favorable for reduction than TeO32. Selectivity of the enzyme for TeO32− decreases with increasing assay pH. Homology modeling of the enzyme identifies four aromatic residues near the active site, including two histidine residues, that are not present in a related SeO32− preferring reductase. On the basis of more favorable π-interactions for Te than for Se and the pH dependence of the selectivity, the Te-selectivity is attributed in part to these aromatic residues. The resulting Te0 enzymatic product resembles Te nanowires.

Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion

Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene (ISG) that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an ISG screen against VSV-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of virus replication. Mechanistically, internalized 25HC accumulates in the late endosomes and blocks cholesterol export, thereby restricting SARS-CoV-2 spike protein catalyzed membrane fusion. Our results highlight a unique antiviral mechanism of 25HC and provide the molecular basis for its possible therapeutic development.

Mechanistic Insights into β-Lactamase-Catalysed Carbapenem Degradation Through Product Characterisation

β-Lactamases are a major threat to the clinical use of carbapenems, which are often antibiotics of last resort. Despite this, the reaction outcomes and mechanisms by which β-lactamases degrade carbapenems are still not fully understood. The carbapenem bicyclic core consists of a β-lactam ring fused to a pyrroline ring. Following β-lactamase-mediated opening of the β-lactam, the pyrroline may interconvert between an enamine (2-pyrroline) form and two epimeric imine (1-pyrroline) forms; previous crystallographic and spectroscopic studies have reported all three of these forms in the contexts of hydrolysis by different β-lactamases. As we show by NMR spectroscopy, the serine β-lactamases (KPC-2, SFC-1, CMY-10, OXA-23, and OXA-48) and metallo-β-lactamases (NDM-1, VIM-1, BcII, CphA, and L1) tested all degrade carbapenems to preferentially give the Δ2 (enamine) and/or (R)-Δ1 (imine) products. Rapid non-enzymatic tautomerisation of the Δ2 product to the (R)-Δ1 product prevents assignment of the nascent enzymatic product by NMR. The observed stereoselectivity implies that carbapenemases control the form of their pyrroline ring intermediate(s)/product(s), thereby preventing pyrroline tautomerisation from inhibiting catalysis.

Glyphosate Determination by Coupling an Immuno-Magnetic Assay with Electrochemical Sensors

Glyphosate (N-(phosphonomethyl)glycine) is the most frequently used broad-spectrum herbicide worldwide. Its mechanism of action is based on the inhibition of an enzyme that is essential to plant growth. Its intensive use has caused global contamination to occur, which has not only affected the ecosystems, but even food and other objects of common use. Thus, there is a pronounced need for developing analytical methods for glyphosate determination in different matrices. Here, an electrochemical competitive immunoassay, based on the use of antibody-modified magnetic particles, has been developed. Tetramethylbenzidine (TMB) has been used as an enzymatic substrate. The extent of the affinity reaction has been achieved by monitoring the current value, due to the reduction of the enzymatic product. A disposable screen-printed electrochemical cell has been used. The calibration curve has been recorded in the 0–10,000 ng/L concentration range, with a detection limit of 5 ng/L and quantification limit of 30 ng/L. The electrochemical immunoassay has also been applied to the analysis of spiked beer samples.

Phosphorescent inner filter effect-based sensing of xanthine oxidase and its inhibitors with Mn-doped ZnS quantum dots

Large bandgap semiconductor ZnS QDs (Mn-doped) were explored for inner filter effect-based sensing of xanthine oxidase and its inhibitors, due to the maximum spectral overlap between the absorption of uric acid (the enzymatic product of xanthine oxidase) and the excitation of Mn-doped ZnS QDs.