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 classes are the major objects of the study: four transhydrogenases, lipoamide dehydrogenase, glutathione reductase, thioredoxin reductase, and mercuric ion reductase; and four dehydrogenase/oxidases, D-amino acid oxidase, L-lactate oxidase, D- aspartate oxidase and glycolate oxidase. Where a specific modification is achieved either chemically or by site directed mutagenesis, the altered enzyme structure and mechanisms is 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 the transhydrogenases are the base(s) which participates in thiol-disulfide interchange, the lysine-glutamate ion pair which may increase the eletrophilicity of the FAD and the nascent thiols produced upon two-electron reduction. In all four transhydrogenases, each nascent thiol has a distinct function - interchange or electron transfer to and from the FAD. In lipoamide dehydrogenase and glutathione reductase we are investigating the acid-base properties of groups in the active site, in the native enzymes and in enzyme in which one of these groups has been modified to remove it from the titration. We are studying, in collaboration, wild type mercuric ion reductase and site specific mutant enzymes in which three pairs of cysteine residues have been changed to Ala or Ser, individually or in pairs. Examples of residues of interest in the dehydrogenases/oxidase are the residue(s) responsible for the extraction of a proton from the substrate alpha-carbon and the residues which stereospecifically position and (and activate) the substrate. The gene from Mycobacterium smegmatis coding for L-lactate oxidase cloned into E. coli has been sequenced and is homologous with glycolate oxidase. The X-ray crystal structure of glycolate oxidase is available. Residues identified by homology or previously shown to be in or near the active site (His and Cys) will be altered and the properties of the mutant enzymes examined. We will modify those residues in glycolate oxidase which appear to be functional in catalysis and determine the properties of the mutant enzymes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Career Awards (K06)
Project #
5K06GM021444-28
Application #
3076346
Study Section
Special Emphasis Panel (NCR)
Project Start
1979-07-01
Project End
1992-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
28
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
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