Substrate-Induced Changes in the Ammonia Channel for Imidazole Glycerol Phosphate Synthase

Abstract

IGP synthase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the unusual nucleotide, N(1)-[(5'-phosphoribulosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) to form 5'-(5-aminoimidazole-4-carboxamide) ribonucleotide (AICAR) and imidazole glycerol phosphate (IGP). A common feature of all glutamine amidotransferases is the upregulation of glutamine hydrolysis in the presence of an acceptor substrate. A refined assay system was developed to establish that Saccharomyces cerevisae IGP synthase shows a 4900-fold stimulation of glutaminase in the presence of the substrate acceptor PRFAR. The structure and function of IGP synthase acceptor substrate binding site were probed with competitive inhibitors that are nucleotide substrate and product analogues. In addition, these analogues were also used to establish that the normal steady-state turnover cycle involves a random sequential mechanism. Upregulation of the glutaminase active site occurs when these competitive inhibitors bind in the nucleotide site over 30 A away. One of the key structural features of IGP synthase is that the transfer of ammonia from the glutaminase site occurs through the (beta/alpha)(8) core of the protein. Upon the basis of the recent substrate-occupied structure for yeast IGP synthase (1), 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 resulting in a conformational switch at the base of the (beta/alpha)(8) core that enables the passage of ammonia through the core of the protein. The overall analysis also leads to further discussion of how the residues that cover the opening of the (beta/alpha)(8) in the closed state may assist the channeling of ammonia at the interface of the two functional domains in the open state.

Altering the allosteric pathway in IGPS suppresses millisecond motions and catalytic activity

“…Thus, differences in the ratios of catalytic efficiencies of the PRFAR-bound and apo enzymes reflect the extent of activation due to the effector ligand (Table 1). These data show that glutaminase activity in WT IGPS is stimulated >4,500-fold in the presence of PRFAR, which is in good agreement with the initial report of 4,900-fold activation by Davisson and coworkers (30). Introduction of single-point mutations in HisF has dramatic effects on PRFAR-induced activation of IGPS, as the V12A mutation reduces glutaminase activity to 70% that of WT, whereas the K19A, V48A, and D98A variants are reduced to ∼3% activity.…”

“…Interestingly, despite very similar chemical shift behavior between the two, K19A IGPS is approximately threefold more active than D98A, consistent with earlier work by Davisson, Wilmanns, and coworkers (30,31) on the K19A and D98A enzymes, respectively. V12A is the only mutation that retains WT-like glutaminase activity with a k cat value that is 50% that of WT IGPS, resulting in a similar reduction in catalytic efficiency.…”

Characterization of the Bifunctional γ-Glutamate-cysteine Ligase/Glutathione Synthetase (GshF) of Pasteurella multocida

“…Tryptophan synthase from S. typhimurium (58,59), thymidylate synthase/dihydrofolate reductase (TS-DHFR) from Leishmania major (48,60), glutamate synthase from Synechocystis sp. (61), and imidazole glycerol-phosphate synthase from yeast (62,63) are very well characterized examples of bifunctional enzymes that exhibit both channeling and interdomain communications. Domaindomain interactions in these enzymes become evident upon formation of substrate-enzyme complexes (or reaction intermediates) at either of the two reaction centers, which reciprocally and allosterically activate turnover at the other reaction center.…”

Asparagine Synthetase Chemotherapy

“…Because all ATP pyrophosphatases convert ATP to AMP, the C-terminal active site of ASNS likely catalyzes activation of the side-chain carboxylate of aspartate to form an electrophilic intermediate, β-aspartyl-AMP (βAspAMP) 1, and inorganic pyrophosphate (PP i ) (Scheme 2) (28,55). As observed in other glutaminedependent amidotransferases (44,46,(56)(57)(58)(59)(60)(61), the two active sites of AS-B are linked by a solvent-inaccessible, intramolecular "tunnel" that is sufficiently wide to allow passage of an ammonia molecule (Figure 1b) (62). Glutamine-dependent asparagine production is therefore accomplished using ammonia as a common intermediate to couple the two "halfreactions" carried out in the independent active sites of the enzyme.…”