Imidazole glycerol phosphate synthase from Thermotoga maritima - Quaternary structure, steady-state kinetics, and reaction mechanism of the bienzyme complex

Imidazole glycerol phosphate synthase, which links histidine and de novo pu rine biosynthesis, is a member of the glutamine amidotransferase family. In bacteria, imidazole glycerol phosphate synthase constitutes a bienzyme com plex of the glutaminase subunit HisH and the synthase subunit HisF. Nascent ammonia produced by HisH reacts at the active site of HisF with N'-((5'-ph osphoribulosyl)formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide to yield the products imidazole glycerol phosphate and 5-aminoimidazole-4-carb oxamide ribotide. In order to elucidate the interactions between HisH and H isF and the catalytic mechanism of the HisF reaction, the enzymes tHisH and tHisF from Thermotoga maritima were produced in Escherichia coil, purified , and characterized. Isolated tHisH showed no detectable glutaminase activi ty but was stimulated by complex formation with tHisF to which either the p roduct imidazole glycerol phosphate or a substrate analogue were bound. Eig ht conserved amino acids at the putative active site of tHisF were exchange d by site-directed mutagenesis, and the purified variants were investigated by steady-state kinetics. Aspartate 11 appeared to be essential for the sy nthase activity both in vitro and in vivo, and aspartate 130 could be parti ally replaced only by glutamate. The carboxylate groups of these residues c ould provide general acid/base catalysis in the proposed catalytic mechanis m of the synthase reaction.