Structure and function of the glutamine phosphoribosylpyrophosphate amidotransferase glutamine site and communication with the phosphoribosylpyrophosphate site

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli exhibits a basal PRPP-independent glutaminase activity having a k(cat)/K(m) that is 0.3% of fully active enzyme. Binding of PRPP activates the enzyme by a structural change that lowers the K(m) for glutamine 100-fold and couples glutamine hydrolysis to synthesis of 5- phosphoribosylamine. By analysis of the x-ray structure of the glutamine site containing bound 6-diazo-5-oxonorleucine, a glutamine affinity analog, and by site-directed mutagenesis we have identified residues important for glutamine binding, catalysis, and coupling with PRPP. Tyr⁷⁴ is a key residue in the coupling between the sites for glutamine in the NH₂-terminal domain and PRPP in the COOH-terminal domain. Arg⁷³ and Asp¹²⁷ have roles in glutamine binding. The x-ray structure indicates that there are no amino acid side chains sufficiently close to Cys¹ to participate as a proton acceptor in formation of the thiolate needed for nucleophilic attack on the carboxamide of glutamine, nor as a general acid for amide nitrogen transfer. Based on the x-ray model of the glutamine site and analysis of a mutant enzyme we propose that the free NH₂ terminus of Cys¹ functions as the proton acceptor and donor. The results indicate that the side chain of Asn¹⁰¹ and the backbone nitrogen of Gly¹⁰² function to stabilize a tetrahedral oxyanion resulting from attack of Cys¹ on the glutamine carboxamide. Cys¹, Arg⁷³, Ash¹⁰¹, Gly¹⁰², and Asp¹²⁷ are conserved in the NH₂-terminal domain of a subfamily of amidotransferases that includes asparagine synthetase, glucosamine 6-phosphate synthase, and glutamate synthase, implying a common function in the four enzymes. Tyr⁷⁴, on the other hand, is conserved only in glutamine PRPP amidotransferase sequences consistent with a specific role in interdomain coupling. The catalytic framework of key glutamine site residues supports the assignment of glutamine PRPP amidotransferase to a recently described Ntn (NH₂-terminal nucleophile) hydrolase family of enzymes.