This application builds on accomplishments made since the last award. During the previous three year funding period, the focus has been primarily on the structural biology of aicar formyltransferase (AICARFT). The gene has been cloned, sequenced, expressed, purified to homogeneity, and the human purH gene product has been crystallized. This is the bifunctional protein which contains both AICARFT and IMPcyclo-hydrolase (inosinicase) activities. Respectively, these are the penultimate and final activities of the purine biosynthesis pathway. In addition, mutagenesis studies have given important insights into the structure of purH, most notably by revealing its arrangement of two closely-linked domains. Furthermore, collaborations with crystallography colleagues have resulted in structure of purH. These confirm the two domain structure of purH, and allow the formulation of structural and mechanistic hypotheses regarding the active sites of the AICARFT and IMPCHase domains. These can now be tested by mutagenesis, inhibitor, and kinetic studies. Specific areas proposed to investigate are: 1. Continued investigation of the structure-function relationships in AICARFT/IMPCH: a) determine the minimal size of the AICARFT and IMPCHase domains by construction of further truncation mutants, b) use of active site directed irreversible inhibitors, and c) site directed mutagenesis based on crystallographic insights; 2. Study of the kinetics and mechanism of AICARFT/IMPCH: a) binding order, b) transient phase kinetics, and c) polyglutamylation effects; 3. Genomic structure of AICARFT/IMPCH; 4. Effect of AICARFT inhibition on cell metabolism.
