Inverse heavy enzyme isotope effects in methylthioadenosine nucleosidases

Heavy enzyme isotope effects occur in proteins substituted with 2H-, 13C-, and 15N-enriched amino acids. Mass alterations perturb femtosecond protein motions and have been used to study the linkage between fast motions and transition-state barrier crossing. Heavy enzymes typically show slower rates for their chemical steps. Heavy bacterial methylthioadenosine nucleosidases (MTANs from Helicobactor pylori and Escherichia coli) gave normal isotope effects in steady-state kinetics, with slower rates for the heavy enzymes. However, both enzymes revealed rare inverse isotope effects on their chemical steps, with faster chemical steps in the heavy enzymes. Computational transition-path sampling studies of H. pylori and E. coli MTANs indicated closer enzyme–reactant interactions in the heavy MTANs at times near the transition state, resulting in an improved reaction coordinate geometry. Specific catalytic interactions more favorable for heavy MTANs include improved contacts to the catalytic water nucleophile and to the adenine leaving group. Heavy bacterial MTANs depart from other heavy enzymes as slowed vibrational modes from the heavy isotope substitution caused improved barrier-crossing efficiency. Improved sampling frequency and reactant coordinate distances are highlighted as key factors in MTAN transition-state stabilization.

Artificial Intelligence Resolves Kinetic Pathways of Magnesium Binding to RNA

Magnesium is an indispensable cofactor in countless vital processes. In order to understand its functional role, the characterization of the binding pathways to biomolecules such as RNA is crucial. Despite the importance, a molecular description is still lacking since the transition from the water-mediated outer-sphere to the direct inner-sphere conformation is on the millisecond timescale and therefore out of reach for conventional simulation techniques. To fill this gap, we use transition path sampling to resolve the binding pathways and to elucidate the role of the solvent in the reaction. The results reveal that the molecular void provoked by the leaving phosphate oxygen of the RNA is immediately filled by an entering water molecule. In addition, water molecules from the first and second hydration shell couple to the concerted exchange. To capture the intimate solute-solvent coupling, we perform a committor analysis as basis for a machine learning algorithm that derives the optimal deep learning model from thousands of scanned architectures using hyperparameter tuning. The results reveal that the properly optimized deep network architecture recognizes the important solvent structures, extracts the relevant information and predicts the commitment probability with high accuracy. Our results provide a quantitative description of solute-solvent coupling which is ubiquitous for kosmotropic ions and governs a large variety of biochemical reactions in aqueous solutions.