Evaluations of tyrosine apodecarboxylase assays for pyridoxal phosphate

The alternate procedures used in the tyrosine apodecarboxylase assays for pyridoxal 5'-phosphate were evaluated to determine optimal conditions. Two preparations of tyrosine apodecarboxylase from Streptococcus faecalis were used: a cell suspension and a partially purified cell-free form. The activity of the decarboxylase was measured in two different assays using [14C]tyrosine or [3H]tyrosine as substrate. The presence of serum proteins caused greater inhibition of the assay for serum pyridoxal phosphate using [14C]tyrosine as substrate than the assay with [3H]tyrosine. In contrast, addition of deproteinized serum extract did not appear to inhibit either assay. The rate of reconstitution of the apodecarboxylase in the cell suspension was at least four times slower than that of the cell-free enzyme. The rate of reconstitution of the cell-free enzyme was faster in acetate than in citrate buffer. Inorganic sulfate or phosphate, at normal plasma concentrations, did not alter either the reconstitution rate of tyrosine decarboxylase or the final activity obtained in the assays using either substrate. The tyrosine apodecarboxylase assay for pyridoxal phosphate can be optimized by using deproteinized sera or plasma and incubating the cell-free apoenzyme with the coenzyme in acetate buffer for a time sufficient to obtain maximum reconstitution.     

Reactions of phosphonate analogs of pyridoxal phosphate with apo-aspartate aminotransferase

We have investigated reactions of the 5-phosphonoethyl and 5-phosphonoethenyl analogs of pyridoxal 5'-phosphate in the coenzyme site of cytosolic aspartate aminotransferase. Acid dissociation constants and equilibrium constants for hydration and for tautomerization have been evaluated for these compounds. In confirmation of previous results, both compounds are partially active. They bind to apoenzyme well and undergo conversion in the presence of glutamate to amine forms which show induced circular dichroism comparable to that of native enzyme. A normal "external" Schiff base is evidently formed with 2-methylaspartate, but the amounts of quinonoid intermediate formed with erythro-3-hydroxyaspartate are less than those formed with pyridoxal phosphate. The pKa of the imine group of the enzyme reconstituted with the phosphonoethyl analog is more than two units lower than that in the native enzyme. Binding of the dicarboxylates glutarate, 2-oxoglutarate, and succinate shifts the pKa upward. The absorption spectra of the resulting complexes indicate the existence of at least three low pH species. A shift of 2.3 to 2.9 ppm to a lower frequency was observed for the 31P NMR signal upon binding of these dicarboxylates or of 2-methylaspartate. Enzyme containing the analogs crystallizes. Polarized absorption spectra suggest that the coenzyme has an orientation similar to that of pyridoxal phosphate in the native enzyme.     

Interaction of pyridoxal phosphate modified cytochromes c with mitoplasts

1. The stability of the native conformation of the heme crevice of pyridoxal phosphate (PLP)-ferricytochromes c as assayed by the pK, for 695 nm absorption band varies considerably. The pKa values are 8.76 for cytochrome c modified by PLP at lysine 79[PLP(Lys 79)-cyt. c], 9.23 for cytochrome c modified by PLP at lysine 86 [PLP(Lys 86)-cyt.c], 9.34 for doubly PLP substituted cytochrome c at lysines 79 and 86 [(PLP)2-cyt. c], 9.50 for triply substituted cytochrome c [(PLP)3-cyt. c] and 9.06 for native cytochrome c, which indicates less stable heme crevice of PLP-cytochrome c. 2. The singly PLP-modified cytochrome c indicate decreased activities with mitochondrial cytochrome c oxidase in the following order: PLP(Lys 86)-cyt. c less than PLP(Lys 79)-cyt. c less than native cytochrome c. The high affinity Km for PLP(Lys 86)-cyt. c, PLP(Lys 79)-cyt. c and native cytochrome c are 0.28 microM, 0.16 microM and 0.02 microM respectively. 3. PLP-cytochromes c show decreased binding affinities to fluorescence probes 12-(9-antroyl)-stearic acid and pyrene-labelled mitoplasts. The quenching of singly PLP-modified cytochrome c depends significantly on the ionic strength.     

The role of Zn2+ in pyridoxal phosphate mediated regulation of glutamic acid decarboxylase in brain

• 1.1. γ-Aminobutyric acid, a major inhibitory neurotransmitter in the CNS, is synthesized by glutamic acid decarboxylase which demonstrates an absolute requirement for pyridoxal phosphate.
• 2.2. At physiological concentrations, zinc stimulates the activity of pyridoxal kinase, enhancing the formation of pyridoxal phosphate, which in turn stimulates the activity of glutamic acid decarboxylase.
• 3.3. At pharmacological concentrations, zinc inhibits the activity of glutamic acid decarboxylase without inhibiting pyridoxal kinase.
• 4.4. These results suggest that zinc may play a role in pyridoxal phosphate-mediated regulation of glutamic acid decarboxylase.

     

Interaction of mitochondrial phosphate carrier with fatty acids and hydrophobic phosphate analogs

Mitochondrial transporters, in particular uncoupling proteins and the ADP/ATP carrier, are known to mediate uniport of anionic fatty acids (FAs), allowing FA cycling which is completed by the passive movement of FAs across the membrane in their protonated form. This study investigated the ability of the mitochondrial phosphate carrier to catalyze such a mechanism and, furthermore, how this putative activity is related to the previously observed HgCl(2)-induced uniport mode. The yeast mitochondrial phosphate carrier was expressed in Escherichia coli and then reconstituted into lipid vesicles. The FA-induced H(+) uniport or Cl(-) uniport were monitored fluorometrically after HgCl(2) addition. These transport activities were further characterized by testing various inhibitors of the two different transport modes. The phosphate carrier was found to mediate FA cycling, which led to H(+) efflux in proteoliposomes. This activity was insensitive to ATP, mersalyl or N-ethylmaleimide and was inhibited by methylenediphosphonate and iminodi(methylenephosphonate), which are new inhibitors of mitochondrial phosphate transport. Also, the HgCl(2) induced Cl(-) uniport mediated by the reconstituted yeast PIC, was found to be inhibited by these reagents. Both methylenediphosphonate and iminodi(methylenephosphonate) blocked unidirectional Cl(-) uptake, whereas Cl(-) efflux was inhibited by iminodi(methylenephosphonate) and phosphonoformic acid only. These results suggest that a hydrophobic domain, interacting with FAs, exists in the mitochondrial phosphate carrier, which is distinct from the phosphate transport pathway. This domain allows for FA anion uniport via the phosphate carrier and consequently, FA cycling that should lead to uncoupling in mitochondria. This might be considered as a side function of this carrier.     

Interaction of pyridoxal phosphate analogues with apoenzymes of gamma-cystathionase and serine sulphhydrase

Comparative studies have been done of the interactions of some coenzyme analogues with the apoenzymes of γ-cystathionase (EC 4.2.1.15) from rat liver and serine sulphhydratase (EC 4.2.1.22) from chicken liver — pyridoxal-phosphate-dependent enzymes catalysing reactions of H₂S release from L-cystein $\text{via}$ α,β-elimination and β-substitution, respectively. It was found that minor modifications (substitutions) in the structure of pyridoxal-5′-phosphate (pyridoxal-P; PLP) result in marked lowering of affinity of the analogues for the apoenzymes. Considerable differences were observed between the various apoenzymes in regard to the mode of their interaction with the pyridoxal-P analogues used.     

Interaction of pyridoxal phosphate with thymidylate synthase: Spectral and equilibrium dialysis studies

• 1.1. Changes in the spectrum of pyridoxal phosphate (PLP) were produced by adding an equimolar amount of native thymidylate synthase, but not by adding denatured enzyme or enzyme modified by sulfhydryl-blocking reagents.
• 2.2. The dissociation constant of the thymidylate synthase-PLP complex determined by equilibrium dialysis was 9 ± 1.6 μM, the maximum number of PLP molecules bound per molecule of native thymidylate synthase was 2.5 ± 0.4, and the Hill coefficient was 0.97.
• 3.3. No evidence of PLP binding was found with denatured thymidylate synthase, and only slight binding was observed when enzyme SH groups were blocked or when the active site was blocked with 5-fluorodeoxyuridylate (FdUMP) and methylenetetrahydrofoliate.
• 4.4. The presence of dUMP, dTMP, or FdUMP interfered with the binding of PLP to thymidylate synthase, and the presence of equimolar amounts of PLP interfered with the binding of dUMP.

     

Pyridoxal phosphate-unrelated inhibition of hippocampal glutamic acid decarboxylase by convulsant pyridoxal sulphate

Previous studies from this laboratory have shown that pyridoxal-5-sulphate, the synthetic analogue of pyridoxal phosphate, causes epileptic seizures including tonic-clonic convulsions. These seizure activities are prevented or reversed by GABA or muscimol. In an attempt to delineate the biochemical basis of these seizure processes further, we have studied and shown that pyridoxal sulphate is a competitive inhibitor of glutamic acid decarboxylase. In addition, the chronic administration of pyridoxal sulphate was shown to reduce the concentration of pyridoxal phosphate in the cerebellum, the cerebrum, and basal ganglion, but not in the hippocampus. The activity of hippocampal glutamic acid decarboxylase was reduced after 1, 3, and 5 days of chronic application of pyridoxal sulphate. The inhibition was demonstrated, whether glutamic acid decarboxylase was assayed in the presence or absence of its coenzyme pyridoxal phosphate. Unlike findings in the hippocampus, the activity of glutamic acid decarboxylase in other brain regions was unaffected following chronic application of pyridoxal sulphate. The selective toxic effects of pyridoxal sulfate to the hippocampus, a brain area well known for its high susceptibility to seizure discharges, deserve additional indepth investigation.     

Interaction of pyridoxal phosphate with the amino groups at the active site of 5- aminolevulinic acid dehydratase in maize

Pyridoxal 5′-phosphate strongly and reversibly inhibited maize leaf 5-amino levulinic acid dehydratase. The inhibition was linearly competitive with respect to the substrate 5-aminolevulinic acid at pH values between 7 to 9.0. Pyridoxal was also effective as an inhibitor of the enzyme but pyridoxamine phosphate was not inhibitory. The results suggest that pyridoxal 5′-phosphate may be interacting with the enzyme either close to or at the 5-aminolevulinic acid binding site. This conclusion was further corroborated by the detection of a Schiff base between the enzyme and the substrate, 5-aminolevulinic acid and by reduction of pyridoxal phosphate and substrate complexes with sodium borohydride