STRUCTURAL STUDIES OF GLUTAMINE AMIDOTRANSFERASES

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The incorporation of nitrogen into biological molecules occurs through the action of a family of enzymes called glutamine amidotransferases. These enzymes transfer NH3 from the side chain of glutamine to an appropriate substrate. There are at least fourteen different glutamine amidotransferases, each catalyzing the transfer of NH3 to a different substrate. Sequence fingerprints have been identified that assign all known glutamine amidotransferase sequences to one of two families. Neither the mechanism of NH3 generation nor the tight coupling between NH3 generation and NH3 transport are understood for any of the enzymes. This important question in biological catalysis is being addressed by determination of three-dimensional structures for three different glutamine amidotransferases in both families by X-ray crystallography. Two of the structure determinations are being done by multiwavelength anomalous diffraction and represent important new applications of this crystallographic methodology. 1. Glutamine PRPP amidotransferase catalyzes the first and committed step in de novo synthesis of purine nucleotides. Defects in the purine pathway have been associated with disorders such as hyperuricemia (gout), immunodeficiency and neurological symptoms. Crystal structures are being determined for the enzyme from both B. subtilis and E. coli in order to elucidate the function of an unusual metal center in B. subtilis and higher eukaryotes that apparently has a novel regulatory function and is not catalytic. 2. GNP synthetase, another enzyme from the purine pathway, is a target of attempts to stop cell growth, specifically tumor cell growth and T-cell proliferation. Crystals of GMP synthetase with substrate and ATP bound and crystals of enzyme alone are an excellent system for structural studies of the coupling of NH3-generating and NH3-transfer activities. 3. Imidazole glycerol 3-phosphate synthase, an enzyme from the histidine pathway, is a heterodimeric glutamine amidotransferase.