University of Illinois Urbana-Champaign

Glutamine Phosphoribosyl Pyrophosphate Amidotransferase From Bacillus Subtilis: Iron-Sulfur Biochemistry and Enzymology

Vollmer, Steven John

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https://hdl.handle.net/2142/70533

Description

Title
Glutamine Phosphoribosyl Pyrophosphate Amidotransferase From Bacillus Subtilis: Iron-Sulfur Biochemistry and Enzymology
Author(s)
Vollmer, Steven John
Issue Date
1983
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 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.
Graduation Semester
1983
Type of Resource
text
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http://hdl.handle.net/2142/70533

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