Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Richard B. Honzatko
Adenylosuccinate synthetase governs the committed step of AMP biosynthesis from IMP: the generation of 6-phosphoryl-IMP from GTP and IMP, followed by the formation of adenylosuccinate from 6-phosphoryl-IMP and L-aspartate. Prokaryotes such as Escherichia coli (E. coli) have a single form of adenylosuccinate synthetase but vertebrates have two isozymes of the synthetase. The basic isozyme, which predominates in muscle, participates putatively in the purine nucleotide cycle, has a higher Km for IMP and exhibits substrate inhibition by this nucleotide. Compared to E. coli structures, there are conformational variations in the IMP pocket both in the ligand-free and IMP-ligated structures of the mouse basic isozyme. Furthermore, IMP has alternative modes of binding to the IMP pocket, and can also bind to the GTP pocket. GDP- and IMP-ligated complexes of the mouse muscle and E. coli systems, which differ only in the type of anion (SO42- Cl - acetate) or cation (Mg2+ or Li+) present in their crystallization milieus, exhibit significant conformational differences in active site loops, consistent with the following two rules: (i) IMP requires a bound anion to the beta-phosphoryl pocket of GTP in order to organize the active site. (ii) Mg2+ may bind preferentially to an active site ligated by both GDP (or GTP) and IMP, and does not bind as a stoichiometric complex of Mg2+/GTP. The feedback inhibitor of the synthetase, AMP, depending on the state of active site ligation, behaves as an analogue of IMP or as an analogue of adenylosuccinate. The mouse basic isozyme complex of adenylosuccinate/GDP/Mg2+/sulfate, reveals significant geometric distortions, tight non-bonded contacts, and a probable state of protonation for adenylosuccinate consistent with the formation of a C-6 purine cation. To a first approximation, adenylosuccinate forms from 6-phosphoryl-IMP and L-aspartate by the movement of the purine ring into a stationary alpha-amino group of L-aspartate.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu
Cristina Valeria Iancu
Iancu, Cristina Valeria, "Conformational variability in adenylosuccinate synthetase as revealed by crystal structures of mouse-muscle and Escherichia coli enzymes " (2002). Retrospective Theses and Dissertations. 1001.