Transcarboxylase is a biotin-containing enzymes from the bacterium, Propionibacterium shermanii. It is but one of many biotin enzymes which are widespread in organisms. They occur in mammals and catalyze important steps in gluconeogenesis, lipogenesis and amino acid metaboslim. In procaryotes, they have been found to generate proton gradients across members. A deficiency of these enzymes in genetic disorders of infants is often fatal. Transcarboxylase is the best characterized of all the biotin enzymes. It is made up of 3 different types of subunits, each of which has a different catalytic function, which is required in the overall reaction. These subunits can be isolated and their individual activity measured in the partial reactions. In addition, the intact active enzyme can be reconstituted from the individual subunits and is fully active. Each subunit has been cloned and its amino acid sequence determined. With the clones as tools, by site-directed mutagenesis, the structures will be altered at specific sites of the individual subunits. The overall aim is to dissect the structure of each subunit and determine the relationship of its structure to its catalytic function in relation to the action of the intact enzyme. The action of biotin is as the carboxyl carrier in biotin enzymes and in all biotin enzymes there is a conserved Val/Ala Met Bct Met surrounding the biotin (Bct is biotinyl lysine). This conservation during millions of years of evolution almost certainly indicates this sequence is essential for the catalysis of biotin enzymes in general, or that it provides the signal that informs the synthetase which attaches the biotin to the lysine, that this is the particular lysine that is to be biotinated posttranslationally.
One aim will be to make changes at this conserved site and others of the biotinyl subunit to determine the exact requirement for the biotin to act as a carboxly carrier and to determine what directs the synthetase to the lysine that is to be biotinated. Site-directed mutagenesis will also be done to determine the functional domains of the other two subunits. To provide further information relating to structure and function, one aim will be to determine the three-dimensional structure of the subunits and mutants of the subunits by X-ray and electron microscopy of their crystals and thus correlate the changes in tertiary structure with changes in functions observed by site- directed mutagenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM040786-04
Application #
3298713
Study Section
Biochemistry Study Section (BIO)
Project Start
1988-09-01
Project End
1993-08-31
Budget Start
1991-09-01
Budget End
1992-08-31
Support Year
4
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
Shenoy, B C; Kumar, G K; Samols, D (1993) Dissection of the biotinyl subunit of transcarboxylase into regions essential for activity and assembly. J Biol Chem 268:2232-8
Thornton, C G; Kumar, G K; Haase, F C et al. (1993) Primary structure of the monomer of the 12S subunit of transcarboxylase as deduced from DNA and characterization of the product expressed in Escherichia coli. J Bacteriol 175:5301-8
Woo, S B; Shenoy, B C; Wood, H G et al. (1993) Effect of deletion from the carboxyl terminus of the 12 S subunit on activity of transcarboxylase. J Biol Chem 268:16413-9
Shenoy, B C; Samols, D; Kumar, G K (1993) The conserved methionines of the 1.3 S biotinyl subunit of transcarboxylase: effect of mutations on conformation and activity. Arch Biochem Biophys 304:359-66
Shenoy, B C; Magner, W J; Kumar, G K et al. (1993) The nonbiotinylated form of the 1.3 s subunit of transcarboxylase binds to avidin (monomeric)-agarose: purification and separation from the biotinylated 1.3 S subunit. Protein Expr Purif 4:85-94
Thornton, C G; Kumar, G K; Shenoy, B C et al. (1993) Primary structure of the 5 S subunit of transcarboxylase as deduced from the genomic DNA sequence. FEBS Lett 330:191-6
Shenoy, B C; Xie, Y; Sha, D et al. (1993) Identification and characterization of a factor which is essential for assembly of transcarboxylase. Biochemistry 32:10750-6
Xie, Y; Shenoy, B C; Magner, W J et al. (1993) Purification and characterization of the recombinant 5 S subunit of transcarboxylase from Escherichia coli. Protein Expr Purif 4:456-64
Shenoy, B C; Xie, Y; Park, V L et al. (1992) The importance of methionine residues for the catalysis of the biotin enzyme, transcarboxylase. Analysis by site-directed mutagenesis. J Biol Chem 267:18407-12