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We have combined equilibrium and steered molecular dynamics (SMD) simulations with principal component and correlation analyses to probe the mechanism of allosteric regulation in imidazole glycerol phosphate (IGP) synthase. An evolutionary analysis of IGP synthase revealed a conserved network of interactions leading from the effector binding site to the glutaminase active site, forming conserved communication pathways between the remote active sites. SMD simulations of the undocking of the ribonucleotide effector N1-[(5'-phosphoribulosyl)-formino]-5'-aminoimidazole carboxamide ribonucleotide (PRFAR) resulted in a large scale hinge-opening motion at the interface. Principal component analysis and a correlation analysis of the equilibration protein motion indicate that the dynamics involved in the allosteric transition are mediated by coupled motion between sites that are more than 25 A apart. Furthermore, conserved residues at the substrate-binding site, within the barrel, and at the interface were found to exhibit highly correlated motion during the allosteric transition. The coupled motion between PRFAR unbinding and the directed opening of the interface is interpreted in combination with kinetic assays for the wild-type and mutant systems to develop a model of allosteric regulation in IGP synthase that is monitored and investigated with atomic resolution.

Imidazole glycerol phosphate (IGP) synthase, a triad glutamine amidotransferase, catalyzes the fifth step in the histidine biosynthetic pathway, where ammonia from glutamine is incorporated into N1-[(5'-phosphoribulosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR) to yield IGP and 5'-(5-aminoimidazole-4-carboxamide) ribonucleotide (AICAR). The triad family of glutamine amidotransferases is formed by the coupling of two disparate subdomains, an acceptor domain and a glutamine hydrolysis domain. Each of the enzymes in this family share a common glutaminase domain for which the glutaminase activity is tightly regulated by an acceptor substrate domain. In IGP synthase the glutaminase and PRFAR binding sites are separated by 30 A. Using kinetic analyses of site-specific mutants and molecular dynamic simulations, we have determined that an interdomain salt bridge in IGP synthase between D359 and K196 (approximately 16 A from the PRFAR binding site) plays a key role in mediating communication between the two active sites. This interdomain contact modulates the glutaminase loop containing the histidine and glutamic acid of the catalytic triad to control glutamine hydrolysis. We propose this to be a general principle of catalytic coupling that may be applied to the entire triad glutamine amidotransferase family.

The metabolic pathway for histidine biosynthesis is interesting from an evolutionary perspective because of the diversity of gene organizations and protein structures involved. Hydrolysis of phosphoribosyl-AMP, the third step in the histidine biosynthetic pathway, is carried out by PR-AMP cyclohydrolase, the product of the hisI gene. The three-dimensional structure of PR-AMP cyclohydrolase from Methanobacterium thermoautotrophicum was solved and refined to 1.7 A resolution. The enzyme is a homodimer. The position of the Zn(2+)-binding site that is essential for catalysis was inferred from the positions of bound Cd(2+) ions, which were part of the crystallization medium. These metal binding sites include three cysteine ligands, two from one monomer and the third from the second monomer. The enzyme remains active when Cd(2+) is substituted for Zn(2+). The likely binding site for Mg(2+), also necessary for activity in a homologous cyclohydrolase, was also inferred from Cd(2+) positions and is comprised of aspartic acid side chains. The putative substrate-binding cleft is formed at the interface between the two monomers of the dimer. This fact, combined with the localization of the Zn(2+)-binding site, indicates that the enzyme is an obligate dimer.

In the complex pathway of histidine biosynthesis, a key branch point linking amino acid and purine biosynthesis is catalyzed by the bifunctional enzyme imidazole glycerol phosphate (IGP) synthase. The first domain of IGP synthase, a triad glutamine amidotransferase, hydrolyzes glutamine to form glutamate and ammonia. Its activity is tightly regulated by the binding of the substrate PRFAR to its partner synthase domain. Recent crystal structures and molecular dynamics simulations strongly suggest that the synthase domain, a (beta/alpha)(8) barrel protein, mediates the insertion of ammonia and ring formation in IGP by channeling ammonia from one remote active site to the other. Here, we combine both mutagenesis experiments and computational investigations to gain insight into the transfer of ammonia and the mechanism of conduction. We discover an alternate route for the entrance of ammonia into the (beta/alpha)(8) barrel and argue that water acts as both agonist and antagonist to the enzymatic function. Our results indicate that the architecture of the two subdomains, most notably the strict conservation of key residues at the interface and within the (beta/alpha)(8) barrel, has been optimized to allow the efficient passage of ammonia, and not water, between the two remote active sites.

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.

Imidazole glycerol phosphate synthase catalyzes formation of the imidazole ring in histidine biosynthesis. The enzyme is also a glutamine amidotransferase, which produces ammonia in a glutaminase active site and channels it through a 30-A internal tunnel to a cyclase active site. Glutaminase activity is impaired in the resting enzyme, and stimulated by substrate binding in the cyclase active site. The signaling mechanism was investigated in the crystal structure of a ternary complex in which the glutaminase active site was inactivated by a glutamine analogue and the unstable cyclase substrate was cryo-trapped in the active site. The orientation of N(1)-(5'-phosphoribulosyl)-formimino-5-aminoimidazole-4-carboxamide ribonucleotide in the cyclase active site implicates one side of the cyclase domain in signaling to the glutaminase domain. This side of the cyclase domain contains the interdomain hinge. Two interdomain hydrogen bonds, which do not exist in more open forms of the enzyme, are proposed as molecular signals. One hydrogen bond connects the cyclase domain to the substrate analogue in the glutaminase active site. The second hydrogen bond connects to a peptide that forms an oxyanion hole for stabilization of transient negative charge during glutamine hydrolysis. Peptide rearrangement induced by a fully closed domain interface is proposed to activate the glutaminase by unblocking the oxyanion hole. This interpretation is consistent with biochemical results [Myers, R. S., et al., (2003) Biochemistry 42, 7013-7022, the accompanying paper in this issue] and with structures of the free enzyme and a binary complex with a second glutamine analogue.