Proceedings of the National Academy of Sciences | 2021
Inverse heavy enzyme isotope effects in methylthioadenosine nucleosidases
Abstract
Significance Femtosecond catalytic site motions are linked to transition-state formation in enzymatic reactions as a consequence of local dynamic interactions. Isotopic labeling of enzyme proteins with 2H, 13C, and 15N perturbs bond vibrational frequencies, altering catalytic site interactions on the femtosecond timescale. Comparative kinetics of heavy and normal enzymes provide insight into the coupling of fast enzyme motions to the catalytic reaction coordinate. Heavy enzymes usually show slowed chemical steps, interpreted as slowed dynamic modes or desynchronization of motions facilitating transition-state formation. Discovery of natural inverse heavy enzyme kinetic isotope effects reveals new principles of coupled protein dynamics. 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.