Linking specific macromolecular motions to enzyme rate acceleration in the TIM barrelsuperfamily of proteins

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Project Summary Enzymes have evolved complex structures that sequester catalytic sites and achieve rates 1026 times faster than non-catalyzed reactions. Extensive studies of active sites and de novo design efforts have been unable to replicate or fully explain this rate acceleration, implicating that the greater protein structure has a critical role in catalysis. Research over the last few decades has highlighted the importance of local and global motions in enzyme activity, but much remains to be known about the link between distal motions and rate accelerations. The TIM barrel scaffold is the focus of this investigation due to its ubiquity, an estimated 10% of all proteins contain a TIM barrel domain, and because it catalyzes a diverse range of chemical reactions. The objective of this proposal is to provide insight into the specific protein motions that are relevant to catalysis and enzymatic rate enhancement, and that could be relevant to a wide selection of enzymes. The TIM barrel-containing enzyme enolase from Saccharomyces cerevisiae was chosen as a model system for this Research Strategy due to its well-characterized chemistry and importance in human diseases, such as Alzheimer’s disease and ischemia. This proposal has two aims: 1) map the flexibility of native enolase across a temperature gradient using hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), and 2) correlate changes in catalytic efficiency with local motions through the development and study of mutants. In order to investigate the role of local and global motions and what information these flexibilities can impart, motions will be measured and studied as functions of temperature over a 10s - 4h time scale. This temperature-dependent information will be correlated with analogous kinetic data and assessed for potential catalytically-relevant networks and emergent trends. This study is a fundamental investigation into the role of protein motions in catalytic rates, but could have dramatic effects on our understanding of structure-function relationships and catalysis. In turn, these finding will inform and improve de novo enzyme design and bioengineering approaches. Furthermore, a greater understanding of the role of protein motions in enolase could inspire new inhibitor targets or suggest novel methods of modulating enolase activity to address its role in human diseases. This Research Strategy will facilitate the applicant’s transition from synthetic inorganic chemistry into an academic career studying metalloenzymes. The training plan will develop the applicant’s scientific skills in protein biochemistry, bioinformatics, and mechanistic enzymology, and facilitate the applicant’s career path through conferences, workshops, and mentorship. This proposal will be carried out with Professor Judith Klinman at the California Institute for Quantitative Biosciences (QB3) at the University of California, Berkeley. QB3 was established in 2000, as a collaborative, multidisciplinary center of innovation that promotes research in the biological sciences using state of the art technology.