The principal objective of this research on flavoenzymes is to determine the functions of specific amino acid residues in the mechanism and to identify those residues within the primary sequence and tertiary structure of the protein. Examples of two flavoprotein families are the major objects of the study: lipoamide dehydrogenase, glutathione reductase and thioredoxin reductase are transhydrogenases; and L-lactate monooxygenase is a dehydrogenase/oxidase. Where a specific modification is achieved either chemically or by site directed mutagenesis, the altered enzyme structure and mechanism are studied in detail including the steady-state kinetics as well as the rapid reaction kinetics of the reductive and oxidative half- reactions, the stable states of the enzyme in reductive and oxidative titrations, the spectral properties (optical, fluorescence and CD), and in several cases the X-ray crystal structures. Examples of residues under study in lipoamide dehydrogenase and glutathione reductase are the base(s) which participates in thiol-disulfide interchange, the lysine-glutamate ion pair which increases the electrophilicity of the FAD and the nascent thiols produced upon two-electron reduction. In all four transhydrogenases, each nascent thiol ha a distinct function - interchange or electron transfer to and from the FAD. In thioredoxin reductase for which the crystal structure has only recently been solved, the putative base, the nascent thiols and several residues which may stabilize a postulated alternate structure are under study. Conditions which promote the postulated conformational change and being investigated. Examples of residues of interest in L-lactate monooxygenase from Mycobacterium smegmatis and expressed in E. coli are the residue(s) responsible for the extraction of a proton from the substrate alpha-carbon, the residue stabilizing negative charge at the flavin N-1 position and the residues which stereospecifically position (and activate) the substrate.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM021444-30
Application #
2173728
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1974-09-01
Project End
1999-03-31
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
30
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Cheng, Zhiyong; Zhang, Jinfeng; Ballou, David P et al. (2011) Reactivity of thioredoxin as a protein thiol-disulfide oxidoreductase. Chem Rev 111:5768-83