4-aminobutyrate aminotransferase

Author

Doo-Sik Kim

Date of Award

3-1983

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Jorge E. Churchich

Committee Members

Wesley D. Wicks, Emil Schiltz

Abstract

This work reports studies on 4-aminobutyrate aminotransferase (4- aminobutyrate: 2-oxoglutarate aminotransferase, EC 2.6.1.19), a key enzyme in the metabolism of neurotransmitter 4-aminobutyrate (GABA).

The mitochondrial enzyme 4-aminobutyrate aminotransferase from pig brain was purified 4000-fold by a combination of CM-Sephadex, DEAE-Sephadex and hydroxyapatite chromatography. This preparation, which migrates as a single band on 7.5% polyacrylamide gel electrophoresis, gives a specific activity of 20 units/mg protein. The enzyme is a dimeric protein with molecular weight of 100,000 made up of two subunits of identical molecular size. The purified holoenzyme contains 1 mol of pyridoxal-5-phosphate/mol of dimer. The dissociation constant for the cofactor molecule is 1 nM. Upon addition of pyridoxal-5-phosphate, a second cofactor molecule is bound to the holoenzyme with a dissociation constant of 3 μM. However, the enzyme has the same k_cat and K_m for 4-aminobutyrate before and after the binding of the second cofactor molecule.

Chemical modifications of the enzyme were accomplished by using gabaculine (5-amino-l, 3-cyclohexadiene carboxylic acid) and m-carboxyphenyl-pyridoxamine-5-phosphate as probes of the catalytic binding sites. The reaction of the enzyme with gabaculine was studied by observing changes in the absorption spectrum of the bound cofactor and by monitoring loss of catalytic activity. The enzyme is completely inactivated by gabaculine, but the dialyzed inactive sample containing 0.5 mol of gabaculine/mol dimer is fully reconstituted by addition of pyridoxal 5-phosphate. Stopped-flow kinetic studies reveal that gabaculine reacts with the cofactor bound to the aminotransferase with a second-order rate constant of 2.5 x 10³ M^-1 s^-1. Fluorometric titrations of the apoprotein with m-carboxyphenyl-pyridoxamine-5-phosphate show the binding of two moles of the inhibitor/mole of enzyme. The binding process is reversible and the affinity of the apoprotein for the inhibitor is at least 10-fold higher than the affinity for the cofactor. It is postulated that the dimeric enzyme contains two potential active sites per dimer, but the binding site characterized by a weaker affinity constant for pyridoxal 5-phosphate becomes functional only after specific chemical modification of the molecule of cofactor tightly bound to the protein.

4-Aminobutyrate aminotransferase is inactivated by incubation with o-phthalaldehyde at pH 7.4. The reaction of 6 lysyl residues per dimer brings about 90% loss of the aminotransferase activity. The substrate 2-oxoglutarate at concentrations higher than the K_m = 0.1mM affords complete protection against the inactivation. Several lines of experimental evidence indicate that o-phthalaldehyde reacts with lysyl residues other than those involved in the binding of pyridoxal-5-P.

It is postulated that the carboxyl groups of 2-oxoglutarate interact with positively charged lysyl residues located at the catalytic site.

4-Aminobutyrate aminotransferase is inactivated by preincubation with bispyridoxal-5-P (mixing molar ratio, 20:1) at pH 7.0. The reaction with bispyridoxal-5-P under pseudo-first order conditions proceeds at a slow rate (k_obs = 0.03 min^-1).

The extent of chemical modification was determined by measuring the spectroscopic properties of P-pyridoxyl and P-pyridoxine chromophores formed after reduction of the enzyme reacted with P¹P²-bis (5'-pyridoxal) diphosphate. Reduction with NaBH₄ results in the incorporation of approximately 2.1 P-pyridoxyl residues/dimer. Thus, the blocking of 2 lysyl residues/dinner is needed for inactivation of the aminotransferase. The time course of inactivation is significantly affected by variations in the pH of the reaction mixtures. Plots of k_obs versus pH indicate the reaction of the bifunctional reagent with lysyl residues characterized by low pK values (pK = 7.3).

The substrate 2-oxoglutarate (10 mM) affords complete protection against inactivation, whereas pyridoxal-5-P failed to prevent the inactivation of the enzyme by bispyridoxal-5-P. It is postulated that binding of 2-oxoglutarate to lysyl residues is the major factor contributing to stabilization of the catalytic site. Several lines of experimental evidence indicate that inactivation of the aminotransferase cannot be related to dissociation of the cofactor from the catalytic site. The bifunctional reagent bispyridoxal-5-P blocks lysyl residues other than those involved in the binding of the cofactor.

4-Aminobutyrate aminotransferase is cleaved by trypsin yielding enzymatically active species which can be separated from the split off peptides by gel filtration. The shortened enzyme derivative gives one band (95,000 MW) on polyacrylomide gradient gel electrophoresis. Changes in protein conformation induced by tryptic digestion were studied by fluorescence spectroscopy. The native enzyme tagged with 5-iodoacetamidofluorescein yields a rotational relaxation time of 106 nanoseconds, whereas the trypsin digested enzyme gives a rotational relaxation time of 33 nanoseconds. The decrease in rotational relaxation time is attributed to flexibility of the polypeptide chain with enhanced rotational freedom of the probe covalently linked to one thiol group. The reactivity of sulfhydryl groups toward 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) is also affected by the trypsin cleavage. More SH groups (2.5/dimer) become reactive toward DTNB as result of trypsin digestion. Local conformational fluctuations are induced as result of the tryptic cleavage, but the catalytic sites remain intact.

The peptides released from 4-aminobutyrate aminotransferase by the trypsin digestion were analyzed on finger-print, and their amino acid composition was determined. The release of peptides of 5,500 molecular weight can account for the difference in molecular weights between native and trypsin treated enzymes.

These results suggest that the multiple forms of 4-aminobutyrate aminotransferase are the products of proteolytic digestion.

The N-terminal sequence of 16 amino acid residues of 4-aminobutyrate aminotransferase was determined by solid-phase technique. The PTH-amino acids produced in the peptide sequencing indicated that the two subunits were homologous peptide chains.

The tryptic peptide of 20 amino acids containing phosphopyridoxyl-lysine was isolated and purified by a combination of gel filtration and ion exchange chromatgraphy. The sequence of this fragment determined by automated Edman degradation was compared with those of cytosolic and mitochondrial aspartate aminotransferase.

Recommended Citation

Kim, Doo-Sik, "4-aminobutyrate aminotransferase. " PhD diss., University of Tennessee, 1983.
https://trace.tennessee.edu/utk_graddiss/13087