Structural and functional relationships of the enzymes of the 4-aminobutyrate shunt : 4-aminobutyrate amino transferase, succinic semialdehyde dehydrogenase and succinic semialdehyde reductase

Author

William G. Hearl

Date of Award

8-1984

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major Professor

Jorge E. Churchich

Committee Members

Jay Joshi, John Koontz

Abstract

This work reports studies on the interrelationship of three mitochondrial enzymes from pig brain involved in the metabolism of the neurotransmitter 4-aminobutyrate; 4-aminobutyrate aminotransferase (E.G. 2.6.1.19), succinic semialdehyde dehydrogenase (B.C. 1.2.1.24) and succinic semialdehyde reductase (E.G. 1.1.1.2).

4-Aminobutyrate aminotransferase is a dimeric protein with a molecular weight of 100,000 made up of two subunits of identical size. The aminotransferase is dependent on the presence of the cofactor, pyridoxal-5- phosphate (PLP), for enzymatic activity. The holoenzyme contains 1 mol of PLP/mol of dimer with a dissociation constant for the cofactor of 1nM. Upon addition of PLP, a second molecule of cofactor is bound to the holoenzyme with a dissociation constant of 3 µM. However, the enzyme has the same K_cat K_m for 4-aminobutyrate before and after the binding of the second cofactor molecule.

The enzyme is capable of converting ω-amino acids into the corresponding semialdehyde by transamination with 2-oxoglutarate. Although the enzyme is generally recognized for its ability to metabolize 4-aminobutyrate, it can also utilize β-alanine and δ-aminovalerate as substrates.

Succinic semialdehyde is the product of the aminotransferase reaction with 4-aminobutyrate as the substrate. The main pathway for this intermediate in the brain is oxidation to succinate catalyzed by succinic semialdehyde dehydrogenase. This enzyme has been shown to be a tetramer, composed of subunits of equal molecular weight of 40,000. The dehydrogenase has a very low K_m for succinic semialdehyde (10 μM) and is sensitive to substrate inhibition by the aldehyde substrate (K_i = 90 μM).

A physical interaction between these two mitochondrial enzymes was investigated using affinity chromatography. Pyridoxal-5-phosphate-Sepharose was prepared and allowed to interact with 4-aminobutyrate aminotransferase. The enzyme was strongly retained by the modified Sepharose when the aminotransferase had a PLP binding site available. 4-Aminobutyrate aminotransferase which had PLP covalently attached at both active sites failed to be retained by PLP-Sepharose.

The immobilized aminotransferase was found to be catalytically competent and was fully characterized kinetically. K_m values of 4.1 mM and 2.0 mM were found for 4-aminobutyrate and 2-oxoglutarate respectively. Both values are several fold higher than the same constants obtained for the enzyme in solution.

The immobilized aminotransferase showed a linear temperature dependence over a range 4 to 40°C, indicating that diffusion does not significantly contribute to the increased extrinsic kinetic parameters seen for the immobilized enzyme. The altered kinetic constants are more likely due to steric effects.

The well defined aminotransferase-Sepharose system was used as an affinity column to detect a possible interaction with succinic semialdehyde dehydrogenase. The dehydrogenase was found to be retained by aminotransferase-Sepharose column at phosphate concentrations below 0.1 M. To preclude the possibility that succinic semialdehyde dehydrogenase was binding to a free PLP moiety by a Schiff's base linkage, the aminotransferase-Sepharose column was reduced by treatment with sodium borohydride. Following the chemical modification, it was found that retained succinic semialdehyde dehydrogenase could be eluted from the column by a linear phosphate gradient at concentrations above 0.1 M.

To reaffirm the presence of a specific aminotransferase-dehydrogenase enzyme complex, antibody was raised against purified 4-aminobutyrate aminotransferase for use in immunoaffinity chromatography. The antibody isolated from immune rabbits was found to be monospecific for aminotransferase. Cross-reactivity with succinic semialdehyde dehydrogenase was not observed by immunodiffusion or by immunoblotting techniques.

Anti-aminotransferase bound to protein A-Sepharose retained both the aminotransferase and succinic semialdehyde dehydrogenase. Elution of the dehydrogenase could be accomplished by treatment of the column with buffers containing greater than 0.2 M sodium chloride. The column retained approximately 1 mol of aminotransferase/1 mol of dehydrogenase.

The immunoaffinity column was also able to retain the enzyme complex from a preparation of mitochondrial proteins. In addition to the detection of succinic semialdehyde dehydrogenase activity on the anti-aminotransferase column, several other unidentified proteins were found to be retained as well.

The interaction between the two mitochondrial enzymes was quantitated using polarization of fluorescence. The polarization of iodoacetamide fluorescein labeled 4-aminobutyrate aminotransferase was shown to increase from 0.121 to 0.219 in the presence of succinic semialdehyde dehydrogenase. A titration of the fluorescein-labeled aminotransferase with the dehydrogenase was performed and a dissociation constant of 0.1 μM was calculated for the enzyme complex.

Recommended Citation

Hearl, William G., "Structural and functional relationships of the enzymes of the 4-aminobutyrate shunt : 4-aminobutyrate amino transferase, succinic semialdehyde dehydrogenase and succinic semialdehyde reductase. " PhD diss., University of Tennessee, 1984.
https://trace.tennessee.edu/utk_graddiss/12881