Enzymes are uniquely able to carry out difficult chemical reactions at high rates. To do this they create a chemical environment in the active site which allows for transition state formation. Frequently, the protein itself contains mobile elements whose motion is essential for active site formation, and coupling of this motion with transition state development is an important problem for the contemporary enzymologist. The transfer of the ribose 5-phosphate group from its universal donor, 5-phosphoribosyl-1-pyrophosphate (PRPP) to specific heterocycles to form nucleotides is carried out by the phosphoribosyltransferases (PRTases). These enzymes are loci of inherited disease, activate anti-tumor prodrugs, and are targets for design of new chemotherapeutics. The PRTases fall into two evolutionary groups, each first identified in the investigator's previous studies. The Type I orotate PRTase (OMP synthase), the major target of this work, follows Rossman fold architecture, and has a prominent peptide loop adjacent to the active site, containing essential catalytic residues. NMR was employed to quantitate the linkage between loop motion and catalytic chemistry and the three dimensional structure of a loop-down form of OMP synthase with all substrates bound has recently been solved. The enzyme undergoes a remarkable reordering of loop and non-loop residues and movement of bound substrates to achieve catalysis. The linkage between loop closing and chemistry is now explored more closely using kinetically characterized mutant OMP synthases altered in each residue of the loop. By determining the three-dimensional structures of mutant OMP synthases, and also of new products and dead-end complexes with wild type, the structural role of the catalytic loop and of substrate movement in catalysis will be revealed. Chemical quench techniques will be employed to follow on-enzyme chemistry, and binding studies to determine the effect of loop alteration on substrate interactions. Kinetic isotope effect (KIE) techniques and positional isotope exchange (PIX) studies will measure proportioning of bound substrates between crossing of the transition state barrier and dissociation to determine how loop closing and formation of the transition state are linked. A second target of the project is the Type II quinolinic acid PRTase, whose general mechanism is poorly understood. The three-dimensional structures of an analog of the initial products complex of wild-type enzyme will be determined as will a complex of PRPP and a quinolinate analog with an Arg 118 mutant in which linkage between binding of the two substrates and between the two subunits has been disrupted. KIE studies will determine the transition state structure of QAPRTase.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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Jones, Warren
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Temple University
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