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Enzymes are nature's catalysts, mediating chemical processes in living systems. The study of enzyme function and mechanism includes defining the maximum catalytic rate and affinity for substrate/s (among other factors), referred to as enzyme kinetics. Enzyme kinetics is a staple of biochemistry curricula and other disciplines, from molecular and cellular biology to pharmacology. However, because enzyme kinetics involves concepts rarely employed in other areas of biology, it can be challenging for students and researchers. Traditional graphical analysis was replaced by computational analysis, requiring another skill not core to many life sciences curricula. Computational analysis can be time-consuming and difficult in free software (e.g., R) or require costly software (e.g., GraphPad Prism). We present Enzyme Kinetics Analysis (EKA), a web-tool to augment teaching and learning and streamline EKA. EKA is an interactive and free tool for analyzing enzyme kinetic data and improving student learning through simulation, built using R and RStudio's ShinyApps. EKA provides kinetic models (Michaelis–Menten, Hill, simple reversible inhibition models, ternary-complex, and ping-pong) for users to fit experimental data, providing graphical results and statistics. Additionally, EKA enables users to input parameters and create data and graphs, to visualize changes to parameters (e.g. KM or number of measurements). This function is designed for students learning kinetics but also for researchers to design experiments. EKA (enzyme-kinetics.shinyapps.io/enzkinet_webpage/) provides a simple, interactive interface for teachers, students, and researchers to explore enzyme kinetics. It gives researchers the ability to design experiments and analyze data without specific software requirements.

Purpose: the COVID-19 pandemic brought unprecedented conditions for overall health care systems by restricting resources for non-COVID-19 patients. As the burden of the disease escalates, routine elective surgeries are being cancelled. The aim of this paper was to provide a guideline for management of endocrine surgical disorders during a pandemic. Methods: we used Delphi method with a nine-scale Likert scale on two rounds of voting involving 64 experienced eminent surgeons and endocrinologists who had the necessary experience to provide insight on endocrine disorder management. All voting was done by email using a standard questionnaire. Results: overall, 37 recommendations were voted on. In two rounds, all recommendations reached an agreement and were either endorsed or rejected. Endorsed statements include dietary change in primary hyperparathyroidism, Cinacalcet treatment in secondary hyperparathyroidism, alpha-blocker administration for pheochromocytoma, methimazole ± β-blocker combination for Graves’ disease, and follow-up for fine-needle aspiration results of thyroid nodules indicated as Bethesda 3–4 cytological results and papillary microcarcinoma. Conclusion: this survey summarizes expert opinion for the management of endocrine surgical conditions during unprecedented times when access to surgical treatment is severely disrupted. The statements are not applicable in circumstances in which surgical treatment is possible.

Bacteriophage-encoded endolysins have been identified as antibacterial candidates. However, the development of endolysins as mainstream antibacterial agents first requires a comprehensive biochemical understanding. This study defines the atomic structure and enzymatic function of Escherichia coli O157:H7 phage FAHEc1 endolysin, LysF1. Bioinformatic analysis suggests this endolysin belongs to the T4 Lysozyme (T4L)-like family of proteins and contains a highly conserved catalytic triad. We then solved the structure of LysF1 with x-ray crystallography to 1.71 Å. LysF1 was confirmed to exist as a monomer in solution by sedimentation velocity experiments. The protein architecture of LysF1 is conserved between T4L and related endolysins. Comparative analysis with related endolysins shows that the spatial orientation of the catalytic triad is conserved, suggesting the catalytic mechanism of peptidoglycan degradation is the same as that of T4L. Differences in the sequence illustrate the role coevolution may have in the evolution of this fold. We also demonstrate that by mutating a single residue within the hydrophobic core, the thermal stability of LysF1 can be increased by 9.4 °C without compromising enzymatic activity. Overall, the characterization of LysF1 provides further insight into the T4L-like class of endolysins. Our study will help advance the development of related endolysins as antibacterial agents, as rational engineering will rely on understanding mutable positions within this protein fold.

N-acetylglucosamine 6-phosphate deacetylase (NagA) catalyzes the conversion of N-acetylglucosamine-6-phosphate to glucosamine-6-phosphate in amino sugar catabolism. This conversion is an essential step in the catabolism of sialic acid in several pathogenic bacteria, including Pasteurella multocida, and thus NagA is identified as a potential drug target. Here, we report the unique structural features of NagA from P. multocida (PmNagA) resolved to 1.95 Å. PmNagA displays an altered quaternary architecture with unique interface interactions compared to its close homolog, the Escherichia coli NagA (EcNagA). We confirmed that the altered quaternary structure is not a crystallographic artifact using single particle electron cryo-microscopy. Analysis of the determined crystal structure reveals a set of hot-spot residues involved in novel interactions at the dimer-dimer interface. PmNagA binds to one Zn2+ ion in the active site and demonstrates kinetic parameters comparable to other bacterial homologs. Kinetic studies reveal that at high substrate concentrations (~10-fold the KM ), the tetrameric PmNagA displays hysteresis similar to its distant neighbor, the dimeric Staphylococcus aureus NagA (SaNagA). Our findings provide key information on structural and functional properties of NagA in P. multocida that could be utilized to design novel antibacterials.

N-Acetylglucosamine-6-phosphate deacetylase (NagA) and glucosamine-6-phosphate deaminase (NagB) are branch point enzymes that direct amino sugars into different pathways. For Staphylococcus aureus NagA, analytical ultracentrifugation and small-angle X-ray scattering data demonstrate that it is an asymmetric dimer in solution. Initial rate experiments show hysteresis, which may be related to pathway regulation, and kinetic parameters similar to other bacterial isozymes. The enzyme binds two Zn2+ ions and is not substrate inhibited, unlike the Escherichia coli isozyme. S. aureus NagB adopts a novel dimeric structure in solution and shows kinetic parameters comparable to other Gram-positive isozymes. In summary, these functional data and solution structures are of use for understanding amino sugar metabolism in S. aureus, and will inform the design of inhibitory molecules.