Chemistry and Regulation of the Oxygen-Dependent Inactivation of Glutamine Phosphoribosyl Pyrophosphate Amidotransferase From Bacillus Subtilis
Bernlohr, David Allen
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https://hdl.handle.net/2142/70510
Description
Title
Chemistry and Regulation of the Oxygen-Dependent Inactivation of Glutamine Phosphoribosyl Pyrophosphate Amidotransferase From Bacillus Subtilis
Author(s)
Bernlohr, David Allen
Issue Date
1982
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
The purpose of the experiments presented was to investigate the chemistry and regulation of the oxygen-dependent inactivation of Bacillus subtilis glutamine P-Rib-PP amidotransferase in vitro and in vivo. A detailed description of the physical and chemical changes occurring in purified amidotransferase has been obtained. Inactivation of the enzyme by oxygen results in oxidation of the essential Fe-S cluster of the enzyme. The sulfide is oxidized to S(DEGREES) bound in a thiocystine linkage and a mixture of unidentified products (presumably SO(,3)('-2) and SO(,4)('-2)). Oxidation of the iron-sulfur center results in a complete loss of all (alpha)-helical structure of the protein. Oxidized amidotransferase aggregates and precipitates readily. A study of the pronounced effects of substrates and allosteric effectors of the enzyme on the rate of its inactivation by oxygen in vitro led to the formulation of a hypothetical model for the regulation of the inactivation in vivo. The combination of the substrates, P-Rib-PP and glutamine, stabilize the enzyme toward oxygen in growing cells. In starving cells, where one or both substrates are predicted to be absent, the inactivation is predicted to be regulated by relative levels of purine ribonucleotides. The levels of the stabilizer (AMP) and destablizers (GMP + GDP + ADP) will determine the rate of oxygen-dependent inactivation. The model was tested by an examination of changes in the intracellular levels of the regulatory molecules under a number of different culture conditions. The model was found not to be consistent with the observed behavior of amidotransferase in vivo. It is not known why amidotransferase is stable in growing cells. The rate of inactivation in starving cells can be regulated by modulation of purine nucleotide levels, but not in a manner consistent with the in vitro kinetic data. The reasons for the noncompliance of the model with in vivo results and speculation dealing with the inactivation is discussed.
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