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Title:Glutamine Phosphoribosyl Pyrophosphate Amidotransferase From Bacillus Subtilis: Iron-Sulfur Biochemistry and Enzymology
Author(s):Vollmer, Steven John
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Chemistry, Biochemistry
Abstract:The iron-sulfur prosthetic group of glutamine PRPP amidotransferase was found to be a {4Fe-4S} cluster. In the native enzyme, the cluster exists in the diamagnetic +2 redox state. Treatment of the cluster with oxidants resulted either in no reaction or in oxidative dissolution of the cluster, i.e., a +3 state was not detected. The cluster was reduced poorly with sodium dithionite, but was reduced readily by photoreduction with 5-deazaflavin. Photoreduction of the native enzyme resulted in formation of the +1 redox state of the cluster. The reduced enzyme was EPR silent. Examination of the reduced enzyme by Mossbauer spectroscopy indicated the presence of multiple unpaired electrons (S (GREATERTHEQ) 3/2). The E(,m) of the +1/+2 couple was determined to be (LESSTHEQ) -620 mV. The reduced enzyme was found to be active in the following assays: glutamine PRPP amidotransferase, glutaminase and PRPP hydrolase; these results demonstrated that the cluster does not have a redox function during catalysis of amide transfer by the enzyme.
The glutamine utilizing site of the enzyme was examined through the use of the affinity ligand 6-diazo-5-oxo-L-norleucine. The thiolate of Cys-1 was identified as the enzyme nucleophile that hydrolyzes glutamine during amide transfer. The complete amino acid sequence of the enzyme is available, and the ligands to the iron-sulfur clusters have been tentatively identified: Cys-382, Cys-434, Cys-437 and Cys-440.
Issue Date:1983
Type:Text
Description:279 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1983.
URI:http://hdl.handle.net/2142/70533
Other Identifier(s):(UMI)AAI8410066
Date Available in IDEALS:2014-12-15
Date Deposited:1983


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