Xenobiotics (drugs, poisons, pollutants, etc.) are metabolized in the human body by a variety of enzyme pathways. The general consequence of these enzymatic reactions is to "detoxify" the xenobiotic and target it for excretion. As organisms are continually exposed to unexpected and diverse chemicals, these enzymatic pathways must be adaptive to assure proper detoxification. Many mechanisms for adaptation exist. For example, expression of some enzyme systems is induced upon exposure to specific molecules. A second mechanism, which operates on the species level, involves genetic diversity in the activities of many competing metabolic pathways. Upon exposure of a population to a novel xenobiotic, variability within the balance of these competing pathways results in heterogeneous metabolism throughout the population. Some individuals will efficiently detoxify and eliminate the xenobiotic, while others may produce metabolites that are more or less toxic than the original compound. This same metabolic diversity complicates the administration of pharmaceutical agents to combat disease, resulting in variable levels of efficacy and toxicity. Ideally, the selection and dosing of individual medications would be specifically tailored to a predicted response within an individual. The scientific study of this genetic diversity and its relation to the administration of pharmaceuticals is the focus of the developing field of pharmacogenetics.
        Our overall goal is to understand the molecular mechanisms by which genetic polymorphisms within the protein sequences of enzymes modulate their relative role in drug metabolism and, consequently, on the variable efficacy and toxicity of administered pharmaceuticals.  Thiopurine S-methyltransferase (TPMT) metabolizes the class of 6-thiopurine medications, including 6-mercaptopurine, 6-thioguanine and azathioprine, and has been extensively investigated over the past few decades. Large variations of TPMT activity exist in humans and a variety of genetic polymorphisms in the TPMT protein sequence have been identified. Approximately 10% of people are heterozygous for one of the polymorphisms, resulting in an intermediate reduction of their cellular TPMT activity. More seriously, 1 in 300 people are homozygous and almost completely enzyme deficient. A clear relationship has been established between these variations in TPMT activity and the risk of life-threatening hematopoietic toxicity as well as the efficacy of 6-thiopurine medications.

        We have determined the three-dimensional structure of TPMT using NMR spectroscopy (figure above) and found it to adopt the topology of the conserved family of SAM-dependent methyltransferases. We have also characterized the consequences of SAM-binding on the conformation and molecular dynamics of the TPMT polypeptide backbone (figure below). Previous research has established that decreased tissue TPMT activities are the result of increased susceptibility of the polymorphic enzymes to proteasomal degradation. We are interested in analyzing the consequences of the polymorphic mutations on the structural and functional properties of TPMT in order to characterize the molecular basis for increased susceptibility to intracellular degradation. We hypothesize that the polymorphisms destabilize the tertiary structure of TPMT and have only minimal effects on their enzymatic activity. Although the structural basis for destabilization has many possibilities, we will specifically distinguish whether the polymorphs adopt an alternative static conformation or, more likely, produce a dynamic and fluctuating structural state of the proteins. Our current results suggest a simple model where an early intermediate in the equilibrium unfolding pathway for TPMT could represent a single destabilized state independently accessed, to varying degrees, by multiple polymorphs under native conditions. In the future, similar studies will be extended to other drug metabolizing enzymes.