Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Richard B. Honzatko
Adenylosuccinate synthetase catalyzes the first committed step in de novo biosyntheses of AMP from IMP and aspartate, using GTP as the energy source. Metal cations, ligand-induced conformational transitions and metabolic effectors significantly influence the catalytic potential of the synthetase. The Mg2+, Mn2+, or Ca 2+ bind to the same site, coordinating alpha-, beta-, and gamma-phosphoryl groups of GTP. The level of catalysis supported by each cation is linked to its influence on the basicity of Asp13, a residue which abstracts the proton from N1 of IMP. Zn2+, a potent inhibitor of the synthetase, coordinates the beta- and gamma-phosphoryl groups of GTP and His41, stabilizing a dead-end complex of the synthetase. The role of metal cations in the synthetase and the related G-proteins contrasts sharply. Hydrogen bond formation between the 5'-phosphate of IMP and Asn38 drives large conformational changes as far as 30 A away. Indeed the 5'-phosphoryl group of IMP, even though it is not directly involves in catalysis, is as important to the stability of the transition state as essential protein side chains directly involved with catalysis. Guanosine 5'diphosphate 3'-diphosphate (ppGpp), a pleiotropic effector of the stringent response, potently inhibits the synthetase. The combination of ppGpp with crystals of the synthetase, however, reveals guanosine 5' -diphosphate 2':3'-cyclic monophosphate, at the GTP Pocket. The sythetase itself may catalyze the formation of the cyclic inhibitor, leading to a tight ligand-enzyme complex. In fact, stringent effectors could modulate synthetase activity by way of several mechanisms, including the formation of a 6-phosphoryl-IMP and cyclic nucleotide complex.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Hou, Zhenglin, "X-ray crystallographic studies on adenylosuccinate synthetase from Escherichia coli " (1998). Retrospective Theses and Dissertations. 11929.