Chemistry of vitamin B6, molecular cloning of its related enzymes and reversible unfolding studies of myo-inositol monophosphatase with Hsc 70

Author

On-Shin Kwon

Date of Award

12-1993

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Jorge E. Churchich

Committee Members

Jayant G. Joshi, Elizabeth E. Howell, Solon Georghiou

Abstract

The main aims of this work are three-fold; first, to study chemical modification of proteins with vitamin B₆ analogues, pyridoxamine or pyridoxamine-5'-phosphate and pyridoxic acid. second, to clone and characterize mammalian genes encoding 4- aminobutyrate aminotransferase (4-aminobutyrate:2-oxoglutarate aminotransferase, E.C. 2.6.1.19) and pyridoxal kinase (ATP: pyridoxal 5'-phosphotransferase, E.C. 2.7.1.35), and third, to study unfolding of myo-inositol monophosphatase and interaction of Hsc 70 with folded and unfolded conformation of the enzyme. In addition, the studies of structual and functional role of pyridoxine (pyridoxamine) 5'-phosphate oxidase (E.C. 1.4.3.5) are also included in appendix.

A general procedure to quantitate the reaction of carbodiimides with carboxyl groups of proteins is described. Pyridoxamine-5'-P reacts with the o-acylisourea intermediate generated during the reaction of carboxyl residues with carbodiimides. The extent of the reaction is determined by measuring the spectroscopic properties, absorption and emission, of P-pyridoxyl residues covalently attached to the proteins. Resolved pig brain aspartate aminotransferase (apoenzyme), inactivated by 1-ethyl-3-(3-dimethylamino propyl) carbodiimide, reacts with [³H] pyridoxamine-5'-P. After trypsin digestion, one peptide labeled with radioactive P-pyridoxyl was separated by reverse phase HPLC.

Luminescence techniques, i.e., fluorescence and phosphorescence, have been employed to study pyridoxic acid bound to proteins through stable amide linkage. Proteins tagged with 4-pyridoxic acid display the following fluorescence properties: (a) emission and excitation spectra centered at around 430 and 320 nm, respectively; (b) fluorescence quantum yields of 0.3-0.4 and (c) average decay times covering the range 8 - 9.6 nanoseconds. The fluorescence properties of the probe have been used to study the dynamics of the protein in the nanosecond time scale. In the absence of molecular oxygen, free and bound 4-pyridoxic acid exhibit long lived emission at room temperature. The long lived emission is red shifted when compared to fluorescence and decays with average life times ranging from 2.2 to 0.6 milliseconds depending on the nature of the protein. The fluorophore pyridoxic acid covalently linked to proteins is suitable to study the dynamics of proteins, i.e., fast and slow motions of the macromolecule in the nanosecond and millisecond time scales, respectively.

4-Aminobutyrate aminotransferase is a key enzyme of the 4-aminobutyric acid shunt. It is responsible for the conversion of the neurotransmitter 4-aminobutyrate to succinic semialdehyde. By using oligonucleotide probes based on partial amino acid sequence data for the pig brain enzyme, several overlapping cDNA clones of 2.0 - 2.2 kilobases in length have been isolated. The largest cDNA clone was selected for sequence analysis. The amino acid sequence predicted from the cDNA sequence shows that the precursor of 4-aminobutyrate aminotransferase consists of the mature enzyme of 473 amino acids residues and an amino-terminal segment of 27 amino acids attributed to the signal peptide. The cofactor pyridoxal-5'-P is bound to lysine residue 330 of the deduced amino acid sequence of the mature enzyme.

Pyridoxal kinase catalyzes the phosphorylation of vitamin B6 (pyridoxal, pyridoxamine, and pyridoxine) using ATP-Zn as phosphoryl donor. By using oligonucleotide probes based on partial amino acid sequence data for the sheep brain enzyme, several candidate cDNA clones have been isolated from porcine brain cDNA library as well as from sheep liver cDNA library.

myo-inositol monophosphatase isolated from pig brain is a very stable dimeric protein characterized by a rotational correlation time of 30 ns. The unfolding and dissociation of the dimeric enzyme (58 kDa) by guanidine hydrochloride have been investigated at equilibrium. The overall process was reversible as judged from the complete recovery of catalytic activity after dilution of guanidine hydrochloride treated samples. Unfolding of myo-inositol monophosphatase was monitored by circular dichroism, fluorescence and steady state emission anisotropy. A folded, monomeric form of the monophosphatase was not detected by the method of denaturant gel filtration. Uncoupled dissociation and unfolding of the oligomeric enzyme could not be demonstrated. The circular dichroism and emission anisotropy results are consistent with a model in which the dimeric protein unfolds in a single cooperative transition from folded dimer to two unfolded monomers.

Fluorescence techniques have been used to investigate the interaction of bovine Hsc 70 with small molecular weight peptides and myo-inositol monophosphatase. The emission properties of Hsc 70 remain invariant upon addition of ATP. The results of steady-state fluorescence indicate that the tryptophan residues of Hsc 70 are exposed to the rapidly relaxing aqueous solvent. Binding of RNase S-peptide to Hsc 70 causes protein fluorescence quenching which was used to determine a dissociation constant k_d = 2.7 uM for the binary Hsc 70 (RNase S-peptide) complex. The octapeptide corresponding to the NH₂ terminal portion of sickle cell hemoglobin recognizes Hsc 70 and binds with a k_d = 9.3uM f. Binding of RNase S-peptide to Hsc 70 produces a small enhancement of ATPase activity. Unfolded myo-inositol monophosphatase, tagged with the fluorescent probe EDANS, recognizes Hsc 70; the formation of a stable complex was detected by steady-state emission anisotropy measurements. The rate and extent of recovery of catalytic activity of unfolded myo-inositol monophosphatase is not influenced by Hsc 70. It is suggested that interaction of Hsc 70 with unfolded proteins in the cell may be able to delay the formation of misfolded structures.

Brain pyridoxine-5'-phosphate oxidase is activated by the tryptophan metabolites 3-hydroxyanthranilate and 3-hydroxykynurenine. 3-hydroxyanthranilate at concentration of 0.03 mM relieves the inhibition elicited by accumulation of the substrate pyridoxine- 5'-P (Ki = 60uM). The results of fluorometric measurements indicate that four molecules of 3-hydroxyanthranilate bind to the dimeric enzyme (56 kDa) with an association constant of 5.5 x 10⁴ M^-1. Differential spectral measurements failed to detect any direct interaction between the cofactor FMN and the effector 3-hydroxyanthranilate. These results are consistant with the hypothesis that the effector molecules bind to sites of the dimeric protein distrinct from the cofactor site. Limited chymotrypsin digestion of pyridoxine-5'-P oxidase yields catalytically active species that are no longer susceptible to activation by 3-hydroxykynurenine. A polypeptide of 16 kDa containing FMN and endowed with full catalytic activity was isolated by ion-exchange chromatography. It is posturated that the structural domain associated with catalytic activity composes approximately one-half of the molecular mass of pyridoxine-5'-P oxidase (28 kDa), whereas the remaining portion of the macromolecule contains regulatory binding sites.

Recommended Citation

Kwon, On-Shin, "Chemistry of vitamin B6, molecular cloning of its related enzymes and reversible unfolding studies of myo-inositol monophosphatase with Hsc 70. " PhD diss., University of Tennessee, 1993.
https://trace.tennessee.edu/utk_graddiss/10712