Our Research Focus
We are studying chemical catalysis with biological systems (enzymes), using the tools of organic and physical chemistry. Chemical catalysis in biological systems is highly stereo- and regio-specific and selective. Most enzymes catalyze reactions at ambient temperature and pressure. These qualities are of great interest for developing new catalysts for organic reactions. Studying enzyme mechanism on the molecular level leads to an in-depth understanding of how evolution uses the principles of chemistry and physics to direct, enhance, and control biological processes. Additionally, understanding how enzymes work can lead to development of new drugs of medical importance, new paths in organic synthesis, and new methodologies in biotechnology.
Enzymes involved in DNA biosynthesis are being studied (mainly TS and DHFR). Many anti-cancer and antibiotic drugs target these enzymes (Figure 1). An interdisciplinary approach is proposed where the techniques of synthetic organic chemistry and molecular biology are used to manipulate substrates and enzymes, respectively. Students in the group will gain knowledge and hands-on experience in organic synthesis, molecular biology and protein purification, structural biology and drug design, enzyme assays and kinetics, isotope effect measurements, and various theoretical aspects of catalysis.
Figure 1. Enzymes involved in DNA synthesis
It has been debated for years whether the motions of an enzyme (dynamics) are important for catalysis. Our research on Thymidylate Synthase (TS) specifically studies how enzyme dynamics influence the kinetics of enzymatic bond cleavage. The reaction mechanism involves two different C-H bond cleavages (steps 4 and 5 in Figure 2). This provides an excellent model system to probe the nature of the activation of different bonds along the catalytic cascade. Experimental methods have been established to explore the nature of both C-H cleavages, using temperature dependency of kinetic isotope effects (KIEs) and site-directed mutagenesis. Various mutants are being studied to examine both local and distal dynamic effects on enzymatic bond activations.
Figure 2. The mechanism of TS catalyzed reaction
Here is a link to the quicktime movie of DHFR - The Movie from the J. Kraut Group at the Univeristy of California at San Diego.
Biosynthesis of the DNA base thymine is required by all organisms and depends on the enzyme thymidylate synthase (TS). For decades it was thought that only one family of thymidlyate synthases existed, however, recently many organisms have been shown to lack the gene coding for this enzyme. This finding has lead to the discovery of an alternative flavin-dependent thymidylate sythase (FDTS), which has been identified in many prokaryotes and viruses, including several severe human pathogens (i.e. anthrax, tuberculosis, typhus, ect.). Our recent findings have shown that the chemical and kinetic mechanism of FDTSs differs greatly from the established mechanism of other TS enzymes. Mechanistic studies and compounds that selectively inhibit FDTS will provide a foundation for antibiotic and antiviral drug design.
One of the most useful models for enzymatic hydride transfers is yeast alcohol dehydrogenase (yADH). YADH catalyzes the oxidation of alcohols to aldehydes using a nicotinamide cofactor and is valuable for studies of transition state structure because the reaction is completely reversible and the hydride transfer is rate-limiting in both directions. Nonetheless, different probes of transition state structure in yADH have yielded contradictory results, some suggesting an early transition state and others a late transition state. We are combining experiment (kinetic isotope effects) and theory (quantum mechanical calculations) in an attempt to develop a model of the transition state that is consistent with our own experiments, as well as those of other groups. Such a model will necessarily require a departure from classical transition state theory in order to account for quantum mechanical behavior of the hydride, especially quantum mechanical tunneling. Preliminary results have been encouraging and the general model we have may find applicability in the other enzymes studied in the Kohen Group.