Subunit interactions and glutamine utilization by Escherichia coli imidazole glycerol phosphate synthase

A selection strategy has been developed to identify amino acid residues inv olved in subunit interactions that coordinate the two half-reactions cataly zed by glutamine amidotransferases, The protein structures known for this c lass of enzymes have revealed that ammonia is shuttled over long distances and that each amidotransferase evolved different molecular tunnels for this purpose. The heterodimeric Escherichia coli imidazole glycerol phosphate ( IGP) synthase was probed to assess if residues in the substrate amination s ubunit (HisF) are critical for the glutaminase activity in the HisH subunit , The activity of the HisH subunit is dependent upon binding of the nucleot ide substrate at the HisF active site. This regulatory function has been ex ploited as a biochemical selection of mutant HisF subunits that retain full activity with ammonia as a substrate but, when constituted as a holoenzyme with wild-type HisH, impair the glutamine-dependent activity of IGP syntha se. The steady-state kinetic constants for these IGP synthases with HisF al leles showed three distinct effects depending upon the site of mutation. Fo r example, mutation of the R5 residue has similar effects on the glutamine- dependent amidotransfer reaction; however, k(cat)/K-m for the glutaminase h alf-reaction was increased 10-fold over that for the wild-type enzyme with nucleotide substrate. This site appears essential for coupling of the gluta mine hydrolysis and ammonia transfer steps and is the first example of a si te remote to the catalytic triad that modulates the process, The results ar e discussed in the context of recent X-ray crystal structures of glutamine amidotransferases that relate the glutamine binding and acceptor binding si tes.