IMIDAZOLE GLYCEROL PHOSPHATE SYNTHASE - THE GLUTAMINE AMIDOTRANSFERASE IN HISTIDINE BIOSYNTHESIS

Two proteins essential for the biosynthesis of the amino acid histidin e in Escherichia coli have been overexpressed and purified to apparent homogeneity. The protein encoded by the hisF gene has an ammonia-depe ndent activity that results in the conversion of the biosynthetic inte rmediate ibulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleo tide (PRFAR) to imidazole glycerol phosphate (IGP) and minoimidazole-4 -carboxamido-1-beta-D-ribofuranosyl 5'-monophosphate (AICAR). The seco nd protein encoded by the hisH gene exhibits no detectable catalytic p roperties with biosynthetic intermediate PRFAR, glutamine, or ammonia. In combination, the proteins are capable of a stoichiometric conversi on of glutamine and PRFAR to form AICAR, IGP, and glutamate. Neither p rotein alone is capable of mediating a conversion of the nucleotide su bstrate to a free metabolic intermediate. The HisH and HisF proteins f orm a stable 1:1 dimeric complex that constitutes the IGP synthase hol oenzyme. Steady-state kinetic parameters for the holoenzyme indicate t hat glutamine is a more efficient substrate relative to ammonium ion b y a factor of 10(3). The HisF subunit will support an ammonia-dependen t reaction with a turnover number similar to that of the holoenzyme wi th glutamine. The glutaminase activity for the holoenzyme is 0.8% of t hat in the presence of the nucleotide substrate PRFAR. There are criti cal subunit interactions that mediate the catalytic properties for glu tamine hydrolysis. The catalytic turnover of glutamine can be increase d up to 37-fold by the addition of either the product IGP or the biosy nthetic precursor mino]-5-aminoimidazole-4-carboxamideribonucleotide ( 5'-ProFAR). The mechanistic significance of this glutaminase activity compared to other trpG type glutamine amidotransferases is discussed.