Inverse heavy enzyme isotope effects in methylthioadenosine nucleosidases

Abstract

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.

Residence Times for Femtomolar and Picomolar Inhibitors of MTANs

“…E. coli and H. pylori MTANs were expressed and purified to >95% homogeneity by gel electrophoresis analysis, as described previously …”

Protein Dynamics and Enzymatic Catalysis

“…In our first attempt, we were able to correct the timing but the overall compression was not strong enough. In a second attempt at design, we were able to make a mutant heavy enzyme that was faster than the light enzyme (an inverse heavy enzyme isotope effect). We made a similar proposal for a mutant form or aromatic amine dehydrogenase, a more complex PCET chemical reaction …”

Perspective: Path Sampling Methods Applied to Enzymatic Catalysis

“…The past 3 years has seen a significant expansion in the application of TPS to understanding enzymatic chemistry. We count 9 separate systems from 6 separate groups ,− being studied in this fashion. The technique is clearly gaining in importance, but there remain basic hurdles to more general application.…”