Understanding the chemical perspectives of enzymes and their medical applications



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

Dihydrofolate Reductase (DHFR)


One of the most intriguing questions in contemporary Enzymology is whether enzyme dynamics evolved to enhance the catalyzed chemical transformation. Dynamics that are coupled to hydrogen quantum mechanical tunneling can now be examined experimentally by monitoring kinetic isotope effects (KIEs) and their temperature dependence. In the current study, dihydrofolate reductase (DHFR), a small monomeric protein that catalyzes a single C-H-C transfer, is used as a model system. Experimental and computational studies have proposed a dynamic network that includes residues in and remote from the active site (Figure 2). We compared the nature of the H-transfer step of the wild-type enzyme (WT), single and double mutants in and out of the active site. The findings so far indicate that the naturally evolved WT DHFR is perfectly tuned for tunneling (ideal donor-acceptor distance and no thermal �gating�). Active site mutations that reduced the size of the hydrophobic group that squeezes the donor and

acceptor together (I to V, A, and G), resulted in longer and less efficient H-transfer distance as evident form kinetic and structural experiments and calculations. Additionally, the dynamics of two remote residues (M42 and G121) where found to be coupled to each other and to the chemistry catalyzed in the active site. These findings support the suggestion that these distal residues synergistically affect the H transfer at the active site of the enzyme.


Here is a link to the quicktime movie of DHFR - The Movie from Sawaya Group in Hiroshima University.

Thymidylate Synthase


Figure 2. The mechanism of TSase catalyzed reactionIt has been debated for years whether the motions of an enzyme (dynamics) are important for catalysis. Our research on Thymidylate Synthase (TSase, or classical TSase) specifically studies how enzyme dynamics influence the kinetics of enzymatic bond cleavage. The reaction mechanism

Figure 3. The mechanism of TSase catalyzed reaction

involves two different C-H bond cleavages (steps 4 and 5 in Figure 3). 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.


Flavin-Dependent Thymidylate Synthase (FDTS)

 Figure 4. The mechanism of FDTS catalyzed reaction

Biosynthesis of the DNA base thymine is required by all organisms and depends on the enzyme thymidylate synthase. 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 TSase enzymes (Figure 4). Mechanistic studies and compounds that selectively inhibit FDTS will provide a foundation for antibiotic and antiviral drug design. 


Alcohol Dehydrogenase (ADH)

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.

Formate Dehydrogenase (FDH)

The role of fast protein structural fluctuations in enzyme-catalyzed reactions is a hotly debated topic in enzymology. Our research on formate dehydrogenase (FDH) is focused on characterizing enzyme active-site motions on the femtosecond to picosecond time scale and their influence on chemical steps in the reaction catalyzed by this enzyme. To directly observe the dynamics on this time scale, novel spectroscopic method, namely two-dimensional infrared spectroscopy (2D IR), are applied. Probing the dynamics of wild-type FDH and site-specific mutants of the hydrophobic residues surrounding the active site (Figure 5) will aid in understanding the correlation between the dynamic motions and kinetic experiments. Thus, 2D IR can be used in conjunction with the

Figure 5. Active site structure of the ternary complex of formate dehydrogenase with azide (blue) and NAD cofactor (magenta).

measurements of kinetic isotope effects (KIEs) methods to

interpret the relevance of the observed dynamics to the functional properties of FDH. The knowledge of protein dynamics could then be used in rational drug design efforts.

Developing Radiopharmaceuticals for P.E.T. imaging

Fast growing cells of a malignant tumor require increased supply of nucleotides for DNA replication. Therefore the activity of thymidylate synthase (TSase) for de novo synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP) is very important for the maintenance of nucleotide pool (see Figure 1 above). This fact makes TSase an attractive target for cancer therapy and tumor imaging. Another equally important fact is the over expressed folate receptors on the tumor cell membrane. Our focus is to develop new radiopharmaceuticals that can be selectively uptaken by the fast growing tumor cells by utilizing the folate receptors and then once inside the cell, radiotraces are trapped by TSase (Figure 6). We are exploring different folates and labeling them by positron-emitting carbon 11 (half-life of ~20 min) that will

 Figure 6. The principle of new developing method for P.E.T. imaging

accumulate metabolically in cancerous cells much better than it would

to normal cells.