Imidazole glycerol phosphate synthase: Structural and kinetic studies of a triad glutamine amidotransferase

Rebecca S Myers, Purdue University

Abstract

Imidazole glycerol phosphate (IGP) synthase is a glutamine amidotransferase (GAT) that incorporates ammonia derived from glutamine into the unusual nucleotide, PRFAR to form AICAR and IGP. A common feature of all GATs is the upregulation of glutamine hydrolysis in the presence of an acceptor substrate. A refined assay system was developed to establish that Saccharomyces cerevisiae IGP synthase shows a 4,900-fold stimulation of glutaminase in the presence of PRFAR. Competitive inhibitors in the PRFAR active site were shown to upregulate the glutaminase active site over 30 Å away. One of the key structural features of IGP synthase is the transfer of ammonia from the glutaminase site through the (β/α)8 core of the protein. A quartet of charged residues (R239, E293, K360 and E465) at the base of the (β/α)8 barrel functions to exclude water while allowing ammonia to pass. MD simulations of R239 mutations demonstrated that the core of the barrel becomes flooded with water occluding the passage of ammonia. Simulations of wild-type IGP synthase demonstrated that K360 undergoes a slight conformational change when ammonia passes the charged quartet then returns to its original conformation. Mutation at K360 allowed ammonia retrograde motion through the plane of the electrostatic quartet. Kinetic investigations of site-directed mutants revealed that a conserved K258 residue is key to productive binding and the overall stoichiometry of the reaction. The binding of the ribulosyl phosphate portion of the substrate PRFAR appears to be transduced through reorientation of K258. We have determined that an interdomain salt bridge between D359 and K196 that is 16 Å from the PRFAR active site is a key interaction between the two active sites. Loss of the aspartate abolished the glutaminase activity. Loss of the lysine resulted in a faster turnover of glutamine and an uncoupling of the two half reactions. In other triad GATs, a similar interdomain contact is seen in the same area; a charged residue from the acceptor domain interacts with residues adjacent to the catalytic triad. Thus, the key interaction we have illustrated in IGP synthase may be a common mechanism of reaction coupling in the triad GATs.

Degree

Ph.D.

Advisors

Davisson, Purdue University.

Subject Area

Biochemistry|Biophysics

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