Design and synthesis of structural and mechanistic probes of Escherichia coli guanosine monophosphate synthetase
Abstract
The last step in the synthesis of GMP is catalyzed by guanosine monophosphate synthetase (GMPS) which requires ATP and a nitrogen equivalent from either glutamine or ammonium salts. To test the hypothesis that amidotransferases like GMPS transfer nitrogen by placing glutamine-hydrolysis and nitrogen-acceptor sites in close proximity, multisubstrate inhibitors were synthesized. These compounds, which incorporate amino acid and nucleotide elements, exhibit high (mM) IC$\sb{50}$ values. This may be because the enzyme binds native substrates in a specific order. Several nucleotide inhibitors of GMPS have been designed and synthesized in collaboration with structural investigations. F-IMP is a hypothesized mimic of the activated form of XMP which is condensed with ATP in the first step of the reaction. N$\sp2$-OH-GMP is the postulated product when hydroxylamine is substituted for glutamine or ammonia as a nitrogen source. While the 6-oxopurine F-IMP does not exhibit tight binding to GMPS, the synthesis of this nucleotide has highlighted the reactivity of the nucleoside. The novel nucleotide N$\sp2$-OH-GMP is synthesized from F-IMP, and represents another example of the use of fluoride displacements from the 2-position of purines. This product inhibits the enzyme in a time-dependent manner. Preliminary progress curves are shown to demonstrate the onset of inhibition. To assist with the ongoing solution of X-ray data, I-ATP was synthesized as a heavy-atom, isomorphous substrate analogue of ATP. This material is a competitive inhibitor of ATP binding, and provided for the phase determination of the diffraction data. A combination of structural data collected with ATP, I-ATP, and kinetic data with the alternate substrate F-ATP, have identified the adenyl binding pocket of GMPS. These studies have led to the development of a working model of GMPS that explains efficient amidotransfer. To accomplish this feat, subtle structural rearrangements follow substrate binding and lead to the potentiation of glutamine hydrolysis and sequestered ammonia transfer.
Degree
Ph.D.
Subject Area
Biochemistry|Molecular biology
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