STRUCTURE AND FUNCTION OF THE GLUTAMINE PHOSPHORIBOSYLPYROPHOSPHATE AMIDOTRANSFERASE GLUTAMINE SITE AND COMMUNICATION WITH THE PHOSPHORIBOSYLPYROPHOSPHATE SITE

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Esc herichia coli exhibits a basal PRPP-independent glutaminase activity h aving a k(cat)/K-m that is 0.3% of fully active enzyme, Binding of PRP P 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 glutami ne 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, Ty r(74) is a key residue in the coupling between the sites for glutamine in the NH2-terminal domain and PRPP in the COOH-terminal domain, Arg( 73) and Asp(127) have roles in glutamine binding, The x-ray structure indicates that there are no amino acid side chains sufficiently close to Cys(1) to participate as a proton acceptor in formation of the thio late needed for nucleophilic attack on the carboxamide of glutamine, n or as a general acid for amide nitrogen transfer, Based on the x-ray m odel of the glutamine site and analysis of a mutant enzyme we propose that the free NH2 terminus of Cys(1) functions as the proton acceptor and donor. The results indicate that the side chain of As-101 and the backbone nitrogen of Gly(102) function to stabilize a tetrahedral oxya nion resulting from attack. of Cys(1) on the glutamine carboxamide. Cy s(1), Arg(73), Asn(101), Gly(102), and Asp(127) are conserved in the N H2-terminal domain of a subfamily of amidotransferases that includes a sparagine synthetase, glucosamine 6-phosphate synthase, and glutamate synthase, implying a common function in the four enzymes. Tyr(74), On the other hand, is conserved only in glutamine PRPP amidotransferase s equences consistent with a specific role in interdomain coupling. The catalytic framework of key glutamine site residues supports the assign ment of glutamine PRPP amidotransferase to a recently described Ntn (N H2-terminal nucleophile) hydrolase family of enzymes.