Fluorescence of cowpea-chlorotic-mottle virus modified with pyridoxal 5'-phosphate

Cowpea chlorotic mottle virus (CCMV), which is stable at pH 5.0, has been modified at this pH with 0.5--0.7 pyridoxal 5'-phosphate molecules per protein subunit. The fluorescence properties of the labelled CCMV protein in different aggregation states of the virus provide information about the labelled part of the protein and the changes induced in its environment, when the nucleo-protein particles are swollen or dissociated. Fluorescence excitation and emission spectra indicate the presence of radiationless energy transfer from the aromatic amino acid residues to the label. Comparison of the fluorescence lifetimes of the labelled and the unlabelled protein confirms the existence of energy transfer. The mobility of the labelled part, which can be estimated from the fluorescence polarization of pyridoxal phosphate chromophore, is higher than expected from the dimensions of the virus and the protein subunits. Polarization values and the fluorescence lifetimes depend on the presence of small amounts of NaCl or MgCl2 in the buffer solution at pH 7.5. This is due to structural changes in the vicinity of the pyridoxal phosphate label of the RNA and of the protein part.     

Fluorometric assay of pyridoxal and pyridoxal 5'-phosphate.

A new and very sensitive fluorometric method for the determination of pyridoxal and pyridoxal 5′-phosphate is reported. The specificity is based on the reductive amination of pyridoxal and its 5′-phosphate with methyl anthranilate and sodium cyanoborohydride at pH 4,5 to 5,0. Separation of the highly fluorescent methyl-N-pyridoxyl anthranilate was achieved by a combination of column and thin-layer chromatography on silica gel. This method has been applied to the assay of pyridoxal and pyridoxal 5′-phosphate in seruum.     

Fluorescence lifetime and anisotropy studies with liver alcohol dehydrogenase and its complexes

From measurements of the apparent phase and modulation fluorescence lifetime of liver alcohol dehydrogenase at multiple modulation frequencies (6, 18, and 30 MHz), the individual lifetimes and fractional intensities of Trp-314 and Trp-15 are calculated. Values of tau 314 = 3.6, tau 15 = 7.3, and f314 = 0.56, at 20 degrees C, are found. These values are in general agreement with values previously reported by Ross et al. [Ross, J.B.A., Schmidt, C.J., & Brand, L. (1981) Biochemistry 20, 4369] using pulse-decay methodology. In ternary complexes formed between the enzyme, NAD+ and either pyrazole or trifluoroethanol, the fluorescence lifetime of Trp-314 is found to be reduced, indicating that the binding of these ligands causes a dynamic quenching of this residue. The lifetime of Trp-314 is decreased more in the trifluoroethanol ternary complex than that with pyrazole. Also, the alkaline quenching transition of alcohol dehydrogenase is found to result in the selective, dynamic quenching of Trp-314. No change in the lifetimes of the two Trp residues is found upon selective removal of the active-site zinc atoms. From studies of the fluorescence anisotropy, r, of the enzyme as a function of added acrylamide (which selectively quenches the surface Trp-15 residue), the steady-state anisotropy of each residue is determined to be r314 = 0.26 and r15 = 0.21. In the ternary complexes the anisotropy of each residue increases slightly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of pyridoxal 5'-phosphate metabolism in liver

The pyridoxal 5′-phosphate content of liver $\text{in vivo}$ and of hepatocytes $\text{in vitro}$ remains unaltered in the presence of excess unphosphorylated vitamin B6 precursors. Studies with isolated hepatocytes and subcellular fractions show that while product inhibition of pyridoxine phosphate oxidase does not limit synthesis sufficiently to account for the phenomenon, inhibition of phosphatase activity produces striking increases in pyridoxal 5′-phosphate concentration. Protein-binding protects it against degradation by the phosphatase. The data suggest that protein-binding and the enzymatic hydrolysis of pyridoxal 5′-phosphate, synthesized in excess, act jointly to preserve the constancy of the cellular content of this coenzyme.     

A novel coenzymatic function of pyridoxal 5'-phosphate

Pyridoxal 5′-phosphate, the vitamin B₆ derivative, acts as the coenzyme of many enzymes involved in amino acid metabolism. Exceptionally, this compound was found covalently bound to glycogen phosphorylase, the key enzyme in the regulation of glycogen metabolism. Although it is essential for the function of phosphorylase, its direct role has remained an enigma. We have recently found that the glucose moiety of pyridoxal (5′)diphospho (1)-α-D -glucose, a conjugate of pyridoxal 5′-phosphate and glucose 1-phosphate through a pyrophosphate linkage, is transferred to the nonreducing end of glycogen, forming a new α-1,4-glucosidic linkage. This finding emphasizes the importance of the direct phosphate-phosphate interaction between the coenzyme and the substrate in the phosphorylase catalytic reaction. We have proposed a catalytic mechanism for phosphorylase in which the phosphate group of pyridoxal 5′-phosphate acts as an electrophile to the phosphate group of glucose 1-phosphate. This appears to represent the first instance of the direct involvement of a phosphate group in catalysis by enzymes.     

Design of pyridoxal 5'-phosphate dependent catalytic antibodies

Modern approaches for developing antibodies with coenzyme-dependent activities are discussed for pyridoxal 5"-phosphate dependent transformation of amino acid as an example. A new type of antigens analogous to enzyme–substrate compounds is suggested for the production of such antibodies. Approaches for the development of pyridoxal antiidiotypic antibody using analogs of coenzyme–substrate compounds and corresponding apoenzyme complexes are reviewed.     

Tight binding of pyridoxal 5'-phosphate to recombinant Escherichia coli pyridoxine 5'-phosphate oxidase

Escherichia coli pyridoxine (pyridoxamine) 5'-phosphate oxidase (PNPOx) catalyzes the oxidation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate to pyridoxal 5'-phosphate (PLP) using flavin mononucleotide (FMN) as the immediate electron acceptor and oxygen as the ultimate electron acceptor. This reaction serves as the terminal step in the de novo biosynthesis of PLP in E. coli. Removal of FMN from the holoenzyme results in a catalytically inactive apoenzyme. PLP molecules bind tightly to both apo- and holoPNPOx with a stoichiometry of one PLP per monomer. The unique spectral property of apoPNPOx-bound PLP suggests a non-Schiff base linkage. HoloPNPOx with tightly bound PLP shows normal catalytic activity, suggesting that the tightly bound PLP is at a noncatalytic site. The tightly bound PLP is readily transferred to aposerine hydroxymethyltransferase in dilute phosphate buffer. However, when the PNPOx. PLP complex was added to aposerine hydroxymethyltransferase suspended in an E. coli extract the rate of reactivation of the apoenzyme was several-fold faster than when free PLP was added. This suggests that PNPOx somehow targets PLP to aposerine hydroxymethyltransferase in vivo.     

Inactivation of rhodanese by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate and other aromatic aldehydes inactivate rhodanese. The inactivation reaches higher extents if the enzyme is in the sulfur-free form. The identification of the reactive residue as an amino group has been made by spectrophotometric determination of the 5'-phosphorylated pyridoxyl derivative of the enzyme. The inactivation increases with pyridoxal 5'-phosphate concentration and can be partially removed by adding thiosulfate or valine. Prolonged dialysis against phosphate buffer also leads to the enzyme reactivation. The absorption spectra of the pyridoxal phosphate - rhodanese complex show a peak at 410 nm related to the Schiff base and a shoulder in the 330 nm region which is probably due to the reaction between pyridoxal 5'-phosphate and both the amino and thiol groups of the enzyme that appear reasonably close to each other. The relationship betweenloss of activity and pyridoxal 5'-phosphate binding to the enzyme shows that complete inactivation is achieved when four lysyl residues are linked to pyridoxal 5'-phosphate.     

Enzymatic dtermination of pyridoxal 5'-phosphate with gamma-cyanoaminobutyric acid aposynthase.

A rapid alternative method is presented for the determination of pyridoxal 5'-phosphate (pyridoxal-P). The method involves the colorimetric analysis of thiocyanate liberated from S-cyanohomocysteine (Hcy (CN)) in the presence of cyanide when catalyzed by the pyridoxal-P dependent enzyme, gamma-cyano-alpha-aminobutyric acid (gamma-CNabu)-synthase (Hcy (CN) thiocyano-lyase [adding CN]). The rate of formation of thiocyanate is determined by the increase in absorbance at 470 nm on treatment of the enzymatic reaction mixture with FeCl3.     

Acetylcholinesterase is not inhibited by pyridoxal 5'-phosphate

Acetylcholinesterase activity was assayed in the absence and presence of pyridoxal 5'-phosphate. If substrate hydrolysis was measured by the pH-stat method, its rate was not significantly affected by pyridoxal 5'-phosphate. In the spectrophotometric assay, however, this compound led to an apparent decrease in rate. The discrepancy between the two assays is explained by stray-light artefacts produced by pyridoxal 5'-phosphate at the wavelengths of the spectrophotometric assay.     

Isotope effect studies of the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii

The pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii shows a nitrogen isotope effect k14/k15 = 0.9770 +/- 0.0021, a carbon isotope effect k12/k13 = 1.0308 +/- 0.0006, and a carbon isotope effect for L-[alpha-2H]histidine of 1.0333 +/- 0.0001 at pH 6.3, 37 degrees C. These results indicate that the overall decarboxylation rate is limited jointly by the rate of Schiff base interchange and by the rate of decarboxylation. Although the observed isotope effects are quite different from those for the analogous glutamate decarboxylase from Escherichia coli [Abell, L. M., & O'Leary, M. H. (1988) Biochemistry 27, 3325], the intrinsic isotope effects for the two enzymes are essentially the same. The difference in observed isotope effects occurs because of a roughly twofold difference in the partitioning of the pyridoxal 5'-phosphate-substrate Schiff base between decarboxylation and Schiff base interchange. The observed nitrogen isotope effect requires that the imine nitrogen in this Schiff base is protonated. Comparison of carbon isotope effects for deuteriated and undeuteriated substrates reveals that the deuterium isotope effect on the decarboxylation step is about 1.20; thus, in the transition state for the decarboxylation step, the carbon-carbon bond is about two-thirds broken.     

Fluorescence lifetime and quenching studies on some interesting diphenylhexatriene membrane probes

The fluorescence lifetimes of a number of membrane probes based on the 1,6-diphenylhexatriene (DPH) chromophore have been measured in small unilamellar phospholipid vesicles and found to be multiphasic. These probes were quenched by sodium iodide with different efficiencies in vesicles and this has been attributed to the depth of the particular probe in the bilayer. The distribution of the probe between the outer and inner monolayer has been determined for those probes with fixed positions in the bilayer. The iodide ion permeability of the bilayer was found to be immeasurably small over a 3 h period.     

Inactivation of rat brain acetylcholinesterase by pyridoxal 5'-phosphate

Effects of pyridoxal 5'-phosphate on the activity of crude and purified acetylcholinesterase from cerebral hemispheres of adult rat brain were examined. Acetylcholinesterase was completely inactivated by incubation with 0.5 mM pyridoxal 5'-phosphate. The enzyme activity remained unaltered in the presence of analogs of pyridoxal 5'-phosphate, pyridoxal, pyridoxamine and pyridoxamine 5'-phosphate. The inhibition of acetylcholinesterase activity by pyridoxal 5'-phosphate appeared to be of a noncompetitive nature, as determined by Lineweaver-Burk analysis. The inhibitory effect of pyridoxal 5'-phosphate on acetylcholinesterase appeared to be a general one, as the activity of the enzyme from the brains of immature chick and egg-laying hen, and from different tissues of the adult male rats, exhibited a similar pattern in the presence of the inhibitor. The inhibitory effects of pyridoxal 5'-phosphate could be reversed upon exhaustive dialysis of the pyridoxal 5'-phosphate-treated acetylcholinesterase preparations. We propose that the effects of pyridoxal 5'-phosphate are due to its interaction with acetylcholinesterase, and that it can be employed as a useful tool for studying biochemical aspects of this important brain enzyme.