4-Aminobutyrate aminotransferase. Conformational changes induced by reduction of pyridoxal 5-phosphate

Conformational changes induced in 4-aminobutyrate aminotransferase (4-aminobutyrate:2-oxoglutarate aminotransferase, EC 2.6.1.19) by conversion of pyridoxal-5-P to pyridoxyl-5-P were examined by two independent methods. The reactivity of the SH groups of the reduced enzyme is increased by chemical modification of the cofactor. 1.8 SH per dimer of modified enzyme react with DTNB, whereas 1.2 SH per dimer of the native enzyme react with the attacking reagent under identical experimental conditions. The modified and native forms of the enzyme bind the fluorescent probe ANS, but the number of binding sites for ANS is increased as result of conversion of P-pyridoxal to P-pyridoxyl. After the conformational changes onset by reduction of the cofactor, the modified enzyme binds one molecule of pyridoxal-5-P with a Kd of 0.1 microM to become catalytically competent. The catalytic site of the reduce enzyme was probed with P-pyridoxal analogs. Like resolved 4-aminobutyrate aminotransferase, the reduced species recognize the phosphorothioate analog and regain 40% of the total enzymatic activity. Since the catalytic parameters of reduced and native 4-aminobutyrate aminotransferase are indistinguishable, it is concluded that the additional catalytic site of the reduced enzyme is functionally identical to that of the native enzyme.     

Nanosecond spectroscopy of a dimeric enzyme: plasma amine oxidase.

The fluorescence dye 1-anilino-naphthalene-8-sulphonic acid (ANS) was used as a probe of non-polar binding sites in the enzyme plasma amine oxidase. Steady fluorescence measurements indicate that ANS binds to a single binding site of the dimeric enzyme with a dissociation constant of 5 microns. This binding site is different from the catalytic binding site. Nanosecond emission anisotropy measurements were performed on the ANS-enzyme in an effort to detect independent rotation of the subunits in the native enzyme. The observed rotational correlation time (phi = 105 ns) corresponds to the rotation of a rigid dimeric macromolecule. A rotational correlation time of 120 ns was obtained with the enzyme labelled with pyrenebutyric acid. It is concluded that the dimeric enzyme does not exhibit any modes of flexibility due to independent rotation of the subunits in the nanosecond range.     

4-Aminobutyrate aminotransferase: identification of lysine residues connected with catalytic activity

Bis-PLP (P'P2-bis[5'-pyridoxal]diphosphate) was used as a probe of the catalytic site of 4-aminobutyrate aminotransferase. It reacts with lysine residues connected with aminotransferase activity and the binding of 1 mol of reduced bis-PLP/enzyme monomer abrogates catalytic activity. The reactive lysine residues are characterized by low pK values (pK = 7.3). The presence of substrate 2-oxoglutarate (4 mM) prevents inactivation of the aminotransferase treated with bis-PLP. After tryptic digestion of the enzyme modified with bis-PLP and reduced with tritiated NaBH4, a radioactive peptide absorbing at 320 nm was separated by reverse-phase high-performance liquid chromatography. The amino acid sequence of the radioactive peptide, elucidated by Edman degradation, revealed that a specific lysine residue of monomeric 4-aminobutyrate aminotransferase has reacted with bis-PLP. The sequence of the modified peptide differs from the sequence of the peptide bearing the cofactor pyridoxal-5-P covalently attached to a lysine residue. It is postulated that the modified lysine residue is involved in direct interactions with negatively charged carboxylic groups of 2-oxoglutarate.     

Brain 4-aminobutyrate aminotransferase. Isolation and sequence of a cDNA encoding the enzyme.

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 acid 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.     

Active site modification of 4-aminobutyrate aminotransferase with ATP analogs

The dialdehyde of oxidized 1,N6-etheno-ATP and adenosine triphosphopyridoxal were used as probes of the catalytic site of 4-aminobutyrate aminotransferase. Both compounds react with lysine residues critically connected with aminotransferase activity. The binding of 1 mol of oxidized 1,N6-etheno-ATP per mol of enzyme or the binding of 1 mol of adenosine triphosphopyridoxal abrogates catalytic activity. The presence of substrate alpha-ketoglutarate (4 mM) prevents inactivation of the aminotransferase by either one of the ATP analogs. Reduction of the enzyme modified with oxidized 1,N6-etheno-ATP yields a chromophore which displays a maximum of emission at 415 nm and a fluorescent lifetime of 21.6 ns. The degree of exposure of the ethenoadenine ring to collisional encounters with the strong quencher KI was determined at pH 7.0. The ethenoadenine ring of the bound ligand is partially shielded from collisional encounters with the quencher. Steady-state emission anisotropy measurements of the bound ligand reveal that oxidized 1,N6-etheno-ATP is not rigidly attached to the protein matrix. It is postulated that the catalytic domain of 4-aminobutyrate aminotransferase is accessible to bulky reagents of greater length than the substrates 4-aminobutyrate and alpha-ketoglutarate.     

Binding of new PLP analogs to the catalytic domain of GABA transaminase

The binding site of Pyridoxal-5-P in 4-aminobutyrate aminotransferase was studied by using analogs of the cofactor. A phosphorothioate analog (PLP(S] recognizes the catalytic site; it forms a stable complex with the apoenzyme (KD = 1nM) and serves as cofactor during catalysis. Replacement of a non-bridged oxygen by sulfur in the phosphate side chain renders a compound which preserves the negative charges needed for correct alignment of the cofactor at the catalytic site. This phosphorothioate analog of PLP can be used to investigate the catalytic site of vitamin B6 dependent enzymes by means of 31P NMR spectroscopy. A bulky P-pyridoxamine derivative, ie, N-4-azido-2-nitrophenyl pyridoxyl-5-P (NANP) competes with natural cofactor for its binding site. Upon illumination, the arylazide of P-pyridoxamine acts as an efficient photolabeling reagent of the protein. A characteristic feature of this photolabeling reagent, ie, its ability to recognize the cofactor binding site, can be exploited to ascertain the chemical nature of amino acid residues at the catalytic site.     

Glutamate decarboxylase side reactions catalyzed by the enzyme

A homogeneous glutamate decarboxylase isolated from pig brain contains 0.8 mol of tightly bound pyridoxal 5-phosphate/enzyme dimer. Upon addition of exogenous pyridoxal 5-phosphate (pyridoxal-5-P), the enzyme acquires maximum catalytic activity. Preincubation of the enzyme with L-glutamate (10 mM) brings about changes in the absorption spectrum of bound pyridoxal-5-P with the concomitant formation of succinic semialdehyde. However, the rate of this slow secondary reaction, i.e. decarboxylative transamination, is 10(-4) times the rate of normal decarboxylation. It is postulated that under physiological conditions enzymatically inactive species of glutamate decarboxylase, generated by the process of decarboxylative transamination, are reconstituted by pyridoxal-5-P produced by the cytosolic enzymes pyridoxal kinase and pyridoxine-5-P oxidase. The catalytic activity of resolved glutamate decarboxylase is recovered by preincubation with phospho-pyridoxyl-ethanolamine phosphate. The experimental evidence is consistent with the interpretation that the resolved enzyme binds the P-pyridoxyl analog, reduces the stability of the covalent bond of the phospho-pyridoxyl moiety, and catalyzes the formation of pyridoxal-5-P.     

Mitochondrial aspartate aminotransferase-independent function of the catalytic binding sites.

The enzyme mitochondrial aspartate aminotransferase from beef liver is a dimer of identical subunits. The enzymatic activity of the resolved enzyme is restored upon addition of the cofactor pyridoxal 5-phosphate. The binding of 1 molecule of cofactor restores 50% of the original enzymatic activity, whereas the binding of a 2nd molecule of cofactor brings about more than 95% recovery of the catalytic activity. Following addition of 1 mol of pyridoxal-5-P per dimer, three forms of the enzyme may exist in solution: apoenzyme-2 pyridoxal 5'-phosphate, apoenzyme-1 pyridoxal 5'-phosphate, and apoenzyme. The enzyme species are separated by affinity chromatography and the following distribution was found: apoenzyme-2 pyridoxal 5'-phosphate/apoenzyme-1 pytidoxal 5'-phosphate/apoenzyme, 2/6/2. Similar distribution was observed after reduction with NaBH4 of the mixture containing apoenzyme and pyridoxal-5-P at a mixing ratio of 1:1. Fluorometric titrations conducted on samples of apoenzyme and apoenzyme-1 pyridoxal 5'-phosphate reveal that the enzyme species display identical affinity towards the inhibitor 4-pyridoxic-5-P (KD equals 1.1 times 10- minus 6 M). It is concluded that the binding of the cofactor to one of the catalytic sites does not affect the affinity of the second site for the inhibitor. These results, obtained by two independent methods, lend strong support to the hypothesis that the two subunits of the enzyme function independently.     

Detection of conformational changes in chloroplast coupling factor 1 by 8-anilino-1-naphthalene-sulphonate fluorescence changes

Chloroplast coupling factor 1 (CF1) contains a high-affinity binding site for 8-anilino-1-napthalene sulphonate (ANS,Kd = 5-6 microM). The binding of ANS to the enzyme is associated with a fluorescence enhancement and a blue-shift in the emission spectrum. ANS only slightly inhibits ATP hydrolysis by CF1. Adenine nucleotides and inorganic phosphate induce a fast ANS fluorescence quenching of about 50% which is due to a decrease in the affinity of the enzyme for ANS (Kd increases from 6 microM to 22 microM) and in the fluorescence quantum yield of the bound probe (by 33%) but not in the number of ANS sites (n = 1). Conversely, Mg and Ca ions induce a fluorescence enhancement of bound ANS. Inactivation of the enzyme enhances ANS fluorescence, eliminates the response to adenine nucleotides and inorganic phosphate but increases the response to divalent metals. The affinity of latent CF1 for ADP (Kd = 12 microM) is considerably higher than for ATP (Kd = 95 microM) in buffer containing EDTA. The Kd for inorganic phosphate is 140 microM. Mg increases the apparent affinity for ATP (Kd = 28 microM) but not for ADP or Pi. Binding of ATP to the tight-sites does not inhibit the ADP or Pi-induced fluorescence quenching but decreases the affinity for ADP (Kd = 34 microM) and for inorganic phosphate (Kd = 320 microM). These results suggest that the ADP and phosphate binding sites are different but not independent from the tight sites. Activation of a Mg-specific ATPase in CF1 by octyl glucoside decreases the affinity for ADP and inorganic phosphate by about threefold but increases the affinity for ATP. ATPase activation of CF1 also increases the Ki for ADP inhibition of ATP hydrolysis. ATPase activation also influences the ANS responses to Ca and Mg. Ca-ATPase activation increases the fluorescence enhancement and the apparent affinity for Ca whereas Mg-ATPase activation specifically increases the Mg-induced fluorescence enhancement. The fluorescence of CF1-bound ANS is enhanced by Dio-9 and quenched by phloridzin, quercetin, Nbf-Cl and FITC. Nbf-Cl and FITC completely inhibit the ADP-induced fluorescence quenching whereas Dio-9 inhibits the Mg-induced fluorescence enhancement. ANS does not relieve the quercetin or phloridzin inhibition of ATP hydrolysis indicating that these inhibitors do not compete with ANS for a common binding site. ANS may be used, therefore, as a sensitive probe to detect conformational changes in CF1 in response to activation or inactivation and to binding of substrates and of inhibitors.     

Synaptic vesicles from hog brain—their isolation and the coupling between synthesis and uptake of γ-aminobutyrate by glutamate decarboxylase

Purified synaptic vesicles were isolated from hog cerebral cortex by a rapid procedure consisting of homogenization of cerebral cortex slices in iso-osmotic sucrose, differential centrifugation and sucrose density-gradient centrifugation. The purity of the vesicles was evaluated both biochemically and morphologically. The vesicles contained high amounts of γ-aminobutyrate (GABA) and acetylcholine at specific concentrations of 390 nmol/mg protein and 7.2 nmol/mg protein respectively.

Glutamate decarboxylase, the enzyme which catalyses GABA formation, binds to the synaptic vesicles in a calcium-dependent manner. The percentage of glutamate decarboxylase bound to the vesicles increases from about 5% without calcium, reaching a plateau of about 60% at 4 mM Ca²⁺. Magnesium in concentrations 0.2–10 mM has no significant effect on glutamate decarboxylase binding. Also in phospholipid vesicles (small unilamellar phosphatidylserine-phosphatidylcholine. 2:1 liposomes) Ca²⁺, but not Mg²⁺, induced the binding of glutamate decarboxylase, reaching a plateau of 50% at 2 mM Ca²⁺. Both in synaptic vesicles and in phospholipid vesicles the calcium-dependent glutamate decarboxylase binding seems to be specific, and not caused by unspecific association of proteins, since the specific binding (bound enzyme activity/mg bound protein) increases 3-fold from 0 to 4 mM Ca²⁺.

The functional role of this binding was studied in GAD containing vesicles by measuring the relationship between the accumulation of [³H]GABA, newly synthetized from [³H]glutamate, and the uptake of added [¹⁴C]GABA. No significant uptake of [¹⁴C]GABA was found under the experimental conditions used, whereas large amounts of [³H]GABA were found within the vesicles. It appears that the [³H]GABA accumulation process is functionally linked to [³H]GABA synthesis and is mediated by the membrane-bound glutamate decarboxylase. This synthesis-coupled uptake of GABA into synaptic vesicles possibly serves to bring about a plasticity effect in previously stimulated GABAergic nerve endings.     

Brain succinic semialdehyde dehydrogenase: identification of reactive lysyl residues labeled with pyridoxal-5'-phosphate

An NAD+ dependent succinic semialdehyde dehydrogenase from bovine brain was inactivated by pyridoxal-5'- phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After NaBH(4) reduction of the pyridoxal-5'-phosphate inactivated enzyme, it was observed that 3.8 mol phosphopyridoxyl residues were incorporated/enzyme tetramer. The coenzyme, NAD+, protected the enzyme against inactivation by pyridoxal-5'-phosphate. The absorption spectrum of the reduced and dialyzed pyridoxal-5'-phosphate-inactivated enzyme showed a characteristic peak at 325 nm, which was absent in the spectrum of the native enzyme. The fluorescence spectrum of the pyridoxyl enzyme differs completely from that of the native enzyme. After tryptic digestion of the enzyme modified with pyridoxal-5'-phosphate followed by [3H]NaBH4 reduction, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. The sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other mammalian succinic semialdehyde dehydrogenase brain species including human. It is suggested that the catalytic function of succinic semialdehyde dehydrogenase is modulated by binding of pyridoxal-5'-phosphate to specific Lys(347) residue at or near the coenzyme-binding site of the protein.     

A Cl and Cl NMR study of chloride binding to the erythrocyte anion transport protein

Band 3, the erythrocyte anion transport protein, mediates the one-for-one exchange of bicarbonate and chloride ions across the membrane and consequently plays an important role in respiration. Binding to the protein forms the first step in the translocation of the chloride across the membrane. ³⁵Cl and ³⁷Cl NMR relaxation measurements at various field strengths were used to study chloride binding to the protein in the presence and absence of the transport inhibitor 4,4′-dinitrostilbene-2,2′-disulfonate. Significant differences occurred in the NMR relaxation rates depending on whether the inhibitor was present or not. The results indicate that the rate of chloride association and dissociation at each external binding site occurs on a time scale of 5 μs. This implies that the transmembrane flux is not limited by the rate of chloride binding to the external chloride binding site of band 3. The rotational correlation-time of chloride bound to band 3 was found to be 20 ns with a quadrupole coupling constant of 3 MHz.     

High affinity receptors for vasoactive intestinal peptide on a human glioma cell line

Vasoactive intestinal peptide (VIP) bound with high affinity (K_d 0.13 nmol/l) to receptors on the human glioma cell line U-343 MG Cl 2:6. The receptors bound the related peptides helodermin. PHM and secretin with 10, 400 and 5000 times lower affinity, respectively. Deamidated VIP (VIP-COOH) and [des-His¹]VIP bound with 10 and 100 times lower affinity. The fragment VIP(7–28) displaced 25% of the receptor-bound ¹²⁵I-VIP whereas VIP(16–28) and VIP(1–22-NH₂) were inactive. The binding of ¹²⁵I-VIP could be completely inhibited by 10 μmol/l of the antagonists [N-Ac-Tyr¹,D-Phe²]GRF(1–29)-NH₂, [pCl-D-Phe⁶,Leu¹⁷]VIP and VIP(10–28); in contrast, the antagonist L-8-K was inactive. Affinity labeling showed that VIP bound to proteins with Mr's of 75 kDa, 66 kDa and 50 kDa, respectively. Following binding, the peptide was rapidly internalized, and at steady-state only 20% of cell-associated ¹²⁵I-VIP was bound to receptors on the cell surface. The internalized ¹²⁵I-VIP was completely degraded to ¹²⁵I-tyrosine which was released from the cells. Degradation of internalized ¹²⁵I-VIP was significantly reduced by chloroquine phenantroline and pepstatin-A. Surface binding and internalization of ¹²⁵I-VIP was increased 3 times by phenantroline, and pepstatin-A caused a 5 times increase in surface binding. Chloroquine reduced surface-bound ¹²⁵I-VIP, but caused retention of internalized ¹²⁵I-VIP.     

The reversible oxidation of vicinal SH groups in 4-aminobutyrate aminotransferase. Probes of conformational changes

4-Aminobutyrate aminotransferase is inactivated by preincubation with iodosobenzoate at pH 7. The reaction of 2 SH residues/dimer resulted in formation of an oligomeric species of Mr = 100,000 detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunits cross-linked via a disulfide bond are dissociated by addition of 2-mercaptoethanol which also restores full catalytic activity (Choi, S. Y., and Churchich, J.E. (1985) J. Biol. Chem. 260, 993-997). The substrate 2-oxoglutarate prevents inactivation of the enzyme by iodosobenzoate and the subsequent formation of one disulfide bond, whereas 4-aminobutyrate has no effect on the reactivity of SH groups with iodosobenzoate. Modified 4-aminobutyrate aminotransferase (containing 1 disulfide bond) catalyzes a half-transamination reaction; but it is unable to react with 2-oxoglutarate to generate the aldimine form of the enzyme. The spectroscopic properties (fluorescence yield and polarization of fluorescence) of PMP bound to the modified enzyme are different from those of pyridoxamine phosphate (PMP) bound to the native enzyme. The polarization of fluorescence values of PMP bound to the cross-linked enzyme, excited over the spectral range 310-370 nm, are greater (25%) than those of the cofactor of the native enzyme. An increase in the polarization values implies that the motion of PMP is restricted when the subunits are cross-linked via a disulfide bond.     

Fluorescent derivatives of the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex.

One sulfhydryl group per polypeptide chain of the pyruvate dehydrogenase component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was selectively labeled with N-[P-(2-benzoxazoyl)phenyl]-maleimide (NBM), 4-dimethylamino-4-magnitude of-maleimidostilbene (NSM), and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) in 0.05 M potassium phosphate (pH 7). Modification of the sulfhydryl group did not alter the enzymatic activity or the binding of 8-anilino-1-naphthalenesulfonate (ANS) or thiochrome diphosphate to the enzyme. The fluorescence of the NBM or NSM coupled to the sulfhydryl group on the enzyme was quenched by binding to the enzyme of the substrate pyruvate the coenzyme thiamine diphosphate, the coenzyme analogue thiochrome diphosphate, the regulatory ligands acetyl-CoA, GTP, and phosphoenolpyruvate, and the acetyl-CoA analogue, ANS. Fluorescence energy transfer measurements were carried out for the enzyme-bound donor-acceptor pairs NBM-ANS, NBM-thiochrome diphosphate ANS-DDPM, and thiochrome diphosphate-DDM. The results indicate that the modified sulfhydryl group is more than 40 A from the active site and approximately 49 A from the acetyl-CoA regulatory site. Thus, a conformational change must accompany the binding of ligands to the regulatory and catalytic sites. Anisotropy depolarization measurements with ANS bound on the isolated pyruvate dehydrogenase in 0.05 M potassium phosphate (pH 7.0) suggest that under these conditions the enzyme is dimeric.     

ADP binding and ATP synthesis by reconstituted H-ATPase from chloroplasts

The H⁺-ATPase from chloroplasts, CF₀F₁, was isolated, purified and reconstituted into asolectin liposomes. The enzyme was brought either into the oxidized state or into the reduced state, and the rate of ATP synthesis was measured after energisation of the proteoliposomes with an acid—base transition ΔpH (pH_in = 5.0, pH_out = 8.5) and a K⁺/valinomycin diffusion potential, Δφ (K⁺_in = 0.6 mM, K⁺_out = 60 mM). A rate of 250 s⁻¹ was observed with the reduced enzyme (85 s⁻¹ in the absence of Δφ). A rate of 50 s⁻¹ was observed with the oxidized enzyme under the same conditions (15 s⁻¹ in the absence of Δφ). The reconstituted enzyme contained 2 ATP_bound per CF₀F₁ and 1 ADP_bound per CF₀F₁. Upon energisation the enzyme was activated and 0.9 ADP per CF₀F₁, was released. Binding of ADP to the active reduced enzyme was observed under different conditions. In the absence of phosphate the rate constant for ADP binding was 10⁵ M⁻¹·s⁻¹ under energized and de-energized conditions. In the presence of phosphate the rate of ADP binding drastically increased under energized conditions, and strongly decreased under de-energized conditions.     

Functions of the 5'-phosphoryl group of pyridoxal 5'-phosphate in phosphorylase: a study using pyridoxal-reconstituted enzyme as a model system

Pyridoxal-reconstituted phosphorylase was used as a model system to study the possible functions of the 5'-phosphoryl group of pyridoxal 5'-phosphate (PLP) in rabbit muscle glycogen phosphorylase. Kinetic study was conducted by using competitive inhibitors of phosphite, an activator, and alpha-D-glucopyranose 1-phosphate (glucose-1-P) to study the relationship between the PLP phosphate and the binding of glucose-1-P to phosphorylase. Fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy of fluorophosphate bound to pyridoxal phosphorylase showed that its ionization state did not change during enzymatic catalysis. Evaluation of the apparent kinetic parameters for the activation of pyridoxal phosphorylase with different analogues having varied pKa2 values demonstrated a dependency of KM on pKa2. Molybdate, capable of binding as chelates in a trigonal-bipyramidal configuration, was tested for its inhibitory property with pyridoxal phosphorylase. On the basis of the results in this study, several conclusions may be drawn: (1) The bound phosphite in pyridoxal phosphorylase and, possibly, the 5'-phosphoryl group of PLP in native phosphorylase do not effect the glucose-1-P binding. (2) One likely function of the 5'-phosphoryl group of PLP in native phosphorylase is acting as an anchoring point to hold the PLP molecule and/or various amino acid side chains in a proper orientation for effective catalysis. (3) The force between the PLP phosphate and its binding site in phosphorylase is mainly electrostatic; a change of ionization state during catalysis is unlikely. (4) Properties of the central atoms of different anions are important for their effects as either activators or inhibitors of pyridoxal phosphorylase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Covalent modification of the solubilized rat liver vitamin K-dependent carboxylase with pyridoxal-5′-phosphate

The carboxylation of the pentapeptide substrate, Phe-Leu-Glu-Glu-Ile, by a rat microsomal vitamin K-dependent carboxylase was stimulated two- to threefold at pyridoxal-5′-P concentrations between 0.5 and 1.0 mm. This stimulation was reduced at concentrations higher than 1.0 mm. The K_m for the pentapeptide was lowered twofold in the presence of 1 mm pyridoxal-5′-P. The activation by pyridoxal-5′-P is specific, as 1 mm pyridoxal, pyridoxine, pyridoxine-5′-P, pyridoxamine, pyridoxamine-5′-P, or 4-pyridoxic acid did not stimulate the pentapeptide carboxylation rate. All six analogs, as well as formaldehyde and acetaldehyde, inhibited the carboxylation reaction in a concentration-dependent manner. The activation of the carboxylase by pyridoxal-5′-P appeared to be mediated by its direct binding to the enzyme via Schiff base formation. Sodium borohydride reduction of solubilized microsomes in the presence of pyridoxal-5′-P, followed by dialysis to remove unbound material, resulted in a carboxylase preparation with a specific activity twice that of the untreated control microsomes. The derivatized enzyme was not further stimulated by added pyridoxal-5′-P. This derivatized carboxylase could be obtained in the absence of pentapeptide and divalent cations. The stimulation of the carboxylase activity by divalent cations and pyridoxal-5′-P was mediated at separate site(s) on the enzyme. Studies of the NH₂-terminal pyridoxalated pentapeptide with both a normal and PLP-modified enzyme, in the presence and absence of PLP, demonstrated competition of the pentapeptide PLP moiety to a PLP site on the enzyme. It was concluded that pyridoxal-5′-P forms a covalent attachment to an ?-NH₂ of a lysine near the active site of the carboxylase.     

Brain pyridoxine-5-phosphate oxidase. Modulation of its catalytic activity by reaction with pyridoxal 5-phosphate and analogs

Pyridoxine-5-P oxidase, the flavoprotein involved in the oxidation of pyridoxamine-5-P and pyridoxine-5-P to pyridoxal-5-P, has been isolated and purified to homogeneity using sheep brain tissues. Inactivation of the oxidase by bis-pyridoxal-5-P results in binding of the inhibitor to specific lysyl residues. After NaBH4 reduction of the inactivated enzyme, it was found that 1 P-pyridoxyl-pyridoxine-P residue was incorporated per enzyme dimer. After trypsin digestion of the bis-PLP modified enzyme, only one peptide absorbing at 320 nm, was separated by reverse-phase high performance liquid chromatography. The amino acid sequence of the labeled peptide was determined by automated Edman degradation. The observations reported in this paper are relevant to the mechanisms underlying the regulation of the catalytic function of pyridoxines-5-P oxidase by the product pyridoxal-5-P. It is postulated that the catalytic function of the oxidase is modulated by binding of pyridoxal-5-P to a specific lysyl residue of the dimeric structure of the protein.     

Probe dependence of correlation times in heme-free extrinsically labeled human hemoglobin

Human heme-free hemoglobin was labeled at beta 93 with either N-iodoacetylaminoethyl-5-naphthalene-1-sulfonate (AEDANS) or fluorescein iodoacetamide (FIA), at beta 1 with pyridoxal-5-phosphate (PLP) and at the heme pocket with anilinonaphthalene-8-sulfonate (ANS). The correlation times associated with these probes ranged from approximately 12 ns for FIA and AEDANS to nearly 20 ns for ANS and PLP. This indicates the presence of internal flexibility in apohemoglobin with librational motions dominated by the mobilities of the monomeric subunits and of the entire dimeric molecules, variously weighted by the different probes. It was not possible to detect motions characterized by correlation times of about 5 ns such as were present in AEDANS-labeled oxyhemoglobin.