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 purine biosynthesis, is a member of the glutamine amidotransferase family. In bacteria, imidazole glycerol phosphate synthase constitutes a bienzyme complex 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'-phosphoribulosyl)formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide to yield the products imidazole glycerol phosphate and 5-aminoimidazole-4-carboxamide ribotide. In order to elucidate the interactions between HisH and HisF and the catalytic mechanism of the HisF reaction, the enzymes tHisH and tHisF from Thermotoga maritima were produced in Escherichia coli, purified, and characterized. Isolated tHisH showed no detectable glutaminase activity but was stimulated by complex formation with tHisF to which either the product imidazole glycerol phosphate or a substrate analogue were bound. Eight conserved amino acids at the putative active site of tHisF were exchanged by site-directed mutagenesis, and the purified variants were investigated by steady-state kinetics. Aspartate 11 appeared to be essential for the synthase activity both in vitro and in vivo, and aspartate 130 could be partially replaced only by glutamate. The carboxylate groups of these residues could provide general acid/base catalysis in the proposed catalytic mechanism of the synthase reaction.
Beismann-Driemeyer
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Universität zu Köln, Institut für Biochemie, Otto-Fischer-Str. 12-14, D-50674 Köln, Germany.
Sterner
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D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers; D007700:Kinetics; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D020124:Thermotoga maritima / Q000201:enzymology*
D017931:DNA Primers; C087227:imidazole glycerol phosphate synthase; D000619:Aminohydrolases