Subunit interactions and glutamine utilization by Escherichia coli imidazole glycerol phosphate synthase.
A selection strategy has been developed to identify amino acid residues involved in subunit interactions that coordinate the two half-reactions catalyzed by glutamine amidotransferases. The protein structures known for this class 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 subunit (HisF) are critical for the glutaminase activity in the HisH subunit. The activity of the HisH subunit is dependent upon binding of the nucleotide substrate at the HisF active site. This regulatory function has been exploited 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 synthase. The steady-state kinetic constants for these IGP synthases with HisF alleles showed three distinct effects depending upon the site of mutation. For example, mutation of the R5 residue has similar effects on the glutamine-dependent amidotransfer reaction; however, k(cat)/K(m) for the glutaminase half-reaction was increased 10-fold over that for the wild-type enzyme with nucleotide substrate. This site appears essential for coupling of the glutamine hydrolysis and ammonia transfer steps and is the first example of a site remote to the catalytic triad that modulates the process. The results are discussed in the context of recent X-ray crystal structures of glutamine amidotransferases that relate the glutamine binding and acceptor binding sites.
Klem
T J
TJ
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907-1333.
Chen
Y
Y
Davisson
V J
VJ
D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000235:genetics / Q000378:metabolism*; D000641:Ammonia / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology*; D005972:Glutaminase / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D021122:Protein Subunits; D012641:Selection, Genetic; D017386:Sequence Homology, Amino Acid
D021122:Protein Subunits; D005973:Glutamine; D000641:Ammonia; C087227:imidazole glycerol phosphate synthase; D005972:Glutaminase; D000619:Aminohydrolases