Doctoral Dissertations

Young Tae Kim

Date of Award

12-1989

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Jorge E. Churchich

Committee Members

Jayant J. Joshit, Daniel M. Roberts, Solon Georghiou

Abstract

This work reports studies of the structural and functional roles of the enzymes: 4-aminobutyrate aminotransferase (4-aminobutyrate : 2-oxoglutarate aminotransferase, E.G. 2.6.1.19), pyridoxal kinase (ATP : pyridoxal 5'-phosphotransferase, E.G. 2.7.1.35), and pyridoxine (pyridoxamine) 5'-phosphate oxidase (E.G. 1.4.3.5.).

Pyridoxal kinase catalyzes the phosphorylation of vitamin B6 (pyridoxal, pyridoxamine, pyridoxine) using ATP-Zn as phosphoryl donor. The enzyme purified from brain tissues is made up of two identical subunits of 40 kDa each.

Physical interactions between pyridoxal kinase and aspartate aminotransferase were detected by means of emission anisotropy and affinity chromatography techniques.

Binding of aspartate aminotransferase (apoenzymes) to pyridoxal kinase tagged with a fluorescent probe (FIT; fluoresceine isothiocyanate) was detected by emission anisotropy measurements at pH 6.8 (150 mM KGl). Upon saturation of the kinase with the aminotransferase, the emission anisotropy increases 22 %. The protein complex is characterized by a dissociation constant of 3 %mu;M.

Time-dependent emission anisotropy measurements conducted with the mixture 5- naphthylamine-1-sulfonic acid-kinase aspartate aminotransferase (apoenzyme), revealed the presence of two rotational correlation times of ø1 = 36 and ø2 = 62 ns. The longer correlation time is attributed to the stable protein complex.

By immobilizing one enzyme (pyridoxal kinase) through interactions with pyridoxal-Sepharose, it was possible to demonstrate that aspartate aminotransferase releases pyridoxal kinase.

A test of compartmentation of pyridoxal-5-phosphate within the protein complex using alkaline phosphatase as trapping agent, indicates that the cofactor generated by the catalytic action of the kinase is channeled to the apotransaminase. The main function of the stable complex formed by the kinase and the aminotransferase is to hinder the release of free pyridoxal-5-phosphate into the bulk solvent.

Chymotrypsin digestion of sheep brain pyridoxal kinase, a dimer of identical subunits each of 40 kDa, yields two fragments of 24 and 16 kDa with concomitant loss of catalytic activity.

The same pattern of digestion was observed when lAF pyridoxal kinase, carrying a fluorescent probe covalently bound to a specific SH residue, was preincubated with chymotrypsin. The kinetics of proteolysis of lAF-pyridoxal kinase was monitored by emission anisotropy; and the analysis of the initial rate of proteolysis at various concentrations of chymotrypsin reveals that the rate of unfolding of native pyridoxal kinase plays an dominant role in the proteolytic process.

The oxidation of pyridoxine-5-P and pyridoxamine-5-P is catalyzed by the cytosolic enzyme pyridoxine-5-P oxidase. This enzyme is a dimer of molecular weight of 56,000 containing 1 mol of FMN.

Chymotryptic digestion of brain pyridoxine-5-P oxidase brings about a 4-fold enhancement of the catalytic power (Vmax and Km) using pyridoxine-5-P as substrate in the assay mixtures. The chymotrypsin-treated enzyme is less susceptible to inhibition by pyridoxal-5-P than the native enzyme.

Fragments arising from limited proteolysis were separated by affinity chromatography using P-pyridoxal-Sepharose as supporting matrix. Catalytically active fractions, eluted by pyridoxine-5-P (5 mM), displayed three bands when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular masses of the three protein bands are considerably lower than 28 kDa, the molecular mass of monomeric pyridoxine-5-P oxidase.

Spectroscopic studies, absorption, fluorescence, and circular dichroism revealed that the microenvironment surrounding the cofactor flavin mononucleotide is not perturbed by limited proteolysis.

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 a molecular weight of 100,000 made up of two subunits of identical size. The aminotransferase is dependent of the presence of the cofactor, pyridoxal-5-phosphate, for enzymatic activity. The holoenzyme contains 1 mole of pyridoxal-5-phosphate/mol of dimer with a dissociation constant for the cofactor of 1 nM. Upon addition of pyridoxal-5-phosphate, a second molecule of cofactor is bound to the holoenzyme with a dissociation constant of 3 μM. However, the enzyme has the same Kcat and Km

Carboxypeptidase A acts on 4-aminobutyrate aminotransferase specifically removing the C-terminal phenylalanine and the amino acid alanine from both of the two subunits.

The carboxypeptidase A digested protein displays Kcat and Km values identical to those of the native enzyme for the substrate 4-aminobutyrate and δ-aminovalerate.

The structural stability of the enzyme, before and after modification, has been investigated by spectroscopic methods and gel permeation techniques. At neutral pH, the absorption spectrum and the emission of tryptophyl residues are not perturbed by cleavage of two amino acids located on the C-terminal portion of the protein. At pH 5.1, the native enzyme preserves its dimeric structure and is able to catalyzes a half transamination reaction upon addition of the substrate 4-aminobutyrate.

In marked contrast to the native enzyme, the modified protein is dissociated at pH 5.1 into monomeric subunits which are catalytically incompetent.

The native enzyme shows a degree of subunit interactions at acid pH not found with the enzyme treated with carboxypeptidase A, suggesting that the amino acids residues (phenylalanine and alanine) play an important role in the structural organization of the two subunits.

Three cysteinyl containing tryptic peptides were isolated and sequenced from mitochondrial 4-aminobutyrate aminotransferase using DABIA (4- dimethylaminoazobenzene-4-iodoacetamide) as specific labeling reagent for sulfhydryl groups. The enzyme is a dimer made up of two identical subunits, but four out of the six cysteinyl residues/dimer form disulfide bonds when treated with iodosobenzoate to yield inactive enzyme species.

To identify the cyteinyl residues undergoing reversible oxidation /reduction, the SDABIA labeling patterns of the fully reduced (active) and fully oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced enzyme contained three labeled peptides. If the enzyme was treated with iodosobenzoate prior to reaction with DABIA and tryptic digestion, only one labeled peptide was detected and identified (peptide I), indicating that the two missing cysteinyl-containing peptides (peptides H, IB) have been oxidized.

The sulfhydryl groups undergoing oxidation/reduction were found to be intersubunit, based on SDS/polyacrylamide gel electrophoresis results. The loss of catalytic activity of 4-aminobutyrate aminotransferase by oxidation of sulfhydryl residues is related to constraints imposed at the subunit interface by the insertion of disulfide bonds.

4-aminobutyrate aminotransferase is inactivated by preincubation with P1P2-bis (5- pyridoxal) diphosphate (bis-PLP) at pH 7.0 The amino acid sequence associated with bis-PLP binding site and tryptophan containing tryptic peptides have been determined.

In order to define the role of specific amino acids in the reaction mechanism and structure of 4-aminobutyrate aminotransferase by applying current molecular biological techniques, a cDNA encoding 4-aminobutyrate aminotransferase was required.

Our approach to isolating cDNA coding from 4-aminobutyrate aminotransferase has been to use antibodies directed against mature aminotransferase to screen a bovine brain λgtl 1 DNA expression library. A recombinant phage containing the cDNA of interest was identified by the immunoreactivity of its cDNA/β-galactosidase fusion protein product after expression in E.coli (Y1090). The fusion protein produced by the clone catalyzes the conversion of 4-aminobutyrate to succinic semialdehyde as demonstrated by continuous spectrophotometric and fluorometric assays.

Purification of the fusion protein was achieved by hydroxyapatite chromatography and affinity chromatography using anti-GABA-T antibodies covalently attached a solid matrix (Affi-Gel 15). The purified fusion protein displays Km values for the substrates, a-ketoglutarate and 4-aminobutyrate, identical to those of the native enzyme.

Immunoblotting experiments revealed that the antibody against 4-aminobutyrate aminotransferase recognizes a fusion protein of approximately 140 kDa.

Of particular note is the enzymatic activity of the fusion protein, which suggests that the cloned GABA-T cDNA must consist of significant amount of GABA-T gene as the cDNA is able to code for catalytically active GABA-T protein product. Apparently the attachment of the GABA-T polypeptide segment to the p-galactosidase polypeptide segment is sufficiently flexible to allow the assembly not only of immunologically detectable domains but also of the active site.

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