Enzymatic Activation of Water

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Living systems use chemical energy derived from the environment to synthesize a large variety of biological polymers; nucleic acids, proteins, polysaccharides and complex glycerolipids. These polymers are not static, but in a constant state of flux, continuously being synthesized, repaired and/or degraded. The degradations of each of these biological polymers are thermodynamically favorable hydrolytic processes. Thus living organisms engage in a Sisyphean struggle to maintain their polymeric structures. Life, as we know it, is possible only because bulk water is an ineffective reactant. A major chemical role of the large number of enzymes that use water to degrade biological molecules is to enhance the reactivity of water in a controlled fashion. Potential chemical mechanisms of water activation, include desolvation, base catalysis and metal ion coordination have been suggested by structural biology and mutagenesis studies, but the contribution of each of these mechanisms remains essentially untested. Kinetic isotope effects are the experimental measurements that provide the most direct insight into transition state structures. Technical difficulties have previously prevented the determination of kinetic isotope effects on water activation, in this research three methods for studying phosphoester hydrolysis that could be readily adapted to other enzymes, including proteases and lipases will be developed. These three methods rely on different advances that have been made in the realm of mass spectrometry. The first two methods take advantage of the coupling of thermal conversion (pyrolysis) to an isotope ratio mass spectrometer. The third method employs whole molecule mass spectrometry and takes advantage of the capabilities of the new generation of quadrupole-time of flight mass spectrometers to precisely determine the isotopic composition of molecules utilizing tandem mass spectrometry. The kinetic isotope effects will enhance our understanding of the mechanism(s) of enzymatic water activation and may lead to improved design of protein and other biomimetic catalysts. When the methodology for determining KIEs is well-developed and tested, further collaborations with other enzymologists will be actively sought to use the special capabilities of the GC/TC/IRMS facility. Analysis of phosphate is relevant not only to enzymology but to a variety of other interdisciplinary areas. Determination of isotopic composition is important in the study of the environment, climate change and archaeology; in addition to their uses as metabolic tracers and tools in the characterization of chemical reaction mechanisms. CWRU has an active biochemistry undergraduate program with ~30 seniors each year engaging in laboratory research projects and participation in summer research programs for women and disadvantaged minorities. This project will increase the awareness of several undergraduates participating in the research in my lab and with collaborators. These individuals, along with the supported graduate student and post-doctoral associate, will be introduced to the capabilities of modern mass spectrometry and how they may be enhanced by the application of stable isotopes. These opportunities should bring a strong and widely applicable analytical skill set to their scientific careers.