Distribution of B6 vitamers in Escherichia coli as determined by enzymatic assay.

An enzymatic method for determination of B6 vitamers is presented. In this method pyridoxal 5'-phosphate is used to activate aposerine hydroxymethyltransferase to form the catalytically active holoenzyme. The active serine hydroxymethyltransferase, and two other enzymes that form a metabolic cycle, convert serine to glycine and CO2 with the concomitant production of two equivalents of NADPH. The rate of the cycle is directly proportional to the amount of active holoserine hydroxymethyltransferase, which is a measure of the amount of pyridoxal 5'-phosphate in the original sample. The cycle operates about 50 times per minute giving a 100-fold enhancement of NADPH production with respect to original pyridoxal 5'-phosphate content. Other B6 vitamers are converted to pyridoxal 5'-phosphate by a preincubation with a combination of pyridoxal kinase and pyridoxine 5'-phosphate oxidase. A complete analysis of B6 vitamers can be completed in less than 1 h and the assay is linear in the 2- to 50-pmol range of pyridoxal 5'-phosphate. The method is applied to the determination of the B6 vitamer pools in extracts of Escherichia coli. The results show that the pool of pyridoxal 5'-phosphate that is not bound to proteins is large enough to account for product inhibition of both pyridoxal kinase and pyridoxine 5'-phosphate oxidase.     

Activator anion binding site in pyridoxal phosphorylase b: the binding of phosphite, phosphate, and fluorophosphate in the crystal.

It has been established that phosphate analogues can activate glycogen phosphorylase reconstituted with pyridoxal in place of the natural cofactor pyridoxal 5'-phosphate (Change YC. McCalmont T, Graves DJ. 1983. Biochemistry 22:4987-4993). Pyridoxal phosphorylase b has been studied by kinetic, ultracentrifugation, and X-ray crystallographic experiments. In solution, the catalytically active species of pyridoxal phosphorylase b adopts a conformation that is more R-state-like than that of native phosphorylase b, but an inactive dimeric species of the enzyme can be stabilized by activator phosphite in combination with the T-state inhibitor glucose. Co-crystals of pyridoxal phosphorylase b complexed with either phosphite, phosphate, or fluorophosphate, the inhibitor glucose, and the weak activator IMP were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the structures of the complexes have been refined to give crystallographic R factors of 18.5-19.2%, for data between 8 and 2.4 A resolution. The anions bind tightly at the catalytic site in a similar but not identical position to that occupied by the cofactor 5'-phosphate group in the native enzyme (phosphorus to phosphorus atoms distance = 1.2 A). The structural results show that the structures of the pyridoxal phosphorylase b-anion-glucose-IMP complexes are overall similar to the glucose complex of native T-state phosphorylase b. Structural comparisons suggest that the bound anions, in the position observed in the crystal, might have a structural role for effective catalysis.     

Relationship between the multiple forms of rat gastric histidine decarboxylase: effects of conditions favouring phosphorylation and dephosphorylation

Conditions favouring protein phosphorylation and dephosphorylation are examined for their effects on activity and charge heterogeneity of the rat gastric mucosal histidine decarboxylase. Incubation of gastric supernatant with various combinations of ATP, Mg2+, cyclic AMP and protein kinase under the blockade of endogenous phosphodiesterase and phosphatase fails to alter significantly enzyme activity as assayed with or without pyridoxal 5'-phosphate. Similar results are found with the purified enzyme. No change occurs in the distribution of activity between the charged forms. In contrast, treatment with alkaline phosphatase both inactivates the enzyme with preservation of heterogeneity, full reactivation being achieved by pyridoxal 5'-phosphate, and reduces the number of forms and converts forms II and III to form I with preservation of the catalytic potentialities. The data suggest that the enzyme heterogeneity may be related in part to the phosphorylation state; the possibility that the gastric enzyme is susceptible to several post-translational modifications is discussed.     

Inactivation of rat gastric mucosal histidine decarboxylase by phosphatase

Histidine decarboxylase of supernatants as well as of purified preparations from rat gastric mucosa is inactivated by a non-specific phosphatase in the absence of pyridoxal 5'-phosphate. The inactivation is a time and concentration-dependent process. Pyridoxal 5'-phosphate, but not histidine, protects the enzyme against phosphatase action. The inactivation is reversible, only pyridoxal 5'-phosphate reactivates the inactivated enzyme. Pyridoxamine 5'-phosphate is ineffective for histidine decarboxylase, but is converted into an active coenzyme only in gastric supernatant. Evidence for the occurrence of an active phosphatase in gastric tissue is also presented; its properties are those of an acid phosphatase and are similar to those of phosphatases hydrolyzing pyridoxal 5'-phosphate in other tissues. The data indicate that phosphatase promotes apoenzyme formation and may play a role in the regulation of histamine synthesis.     

Structural and functional properties of Drosophila melanogaster phosphorylase: comparison with the rabbit skeletal muscle enzyme

Glycogen phosphorylase isolated from Drosophila melanogaster contains one pyridoxal 5'-phosphate per subunit; the coenzyme is in a hydrophobic environment. Fruit-fly phosphorylase a has lower KM for glucose-1-phosphate and is less sensitive to allosteric inhibitors than the b form of the enzyme. The amino acid composition of Drosophila phosphorylase differs from that of rabbit skeletal muscle phosphorylase. These two enzymes give distinct one dimensional peptide maps. The distribution of reactive SH-groups is markedly different in the insect and vertebrate phosphorylase. Fruit-fly phosphorylase a is dephosphorylated by either rabbit or Drosophila protein phosphatase-1 at a slower rate than rabbit muscle phosphorylase a.     

4-Phospho-hydroxy-l-threonine is an obligatory intermediate in pyridoxal 5'-phosphate coenzyme biosynthesis in Escherichia coli K-12

Abstract We show that thrB -encoded homoserine kinase is required for growth of Escherichia coli K-12 pdxB mutants on minimal glucose medium supplemented with 4-hydroxy-l-threonine (synonym, 3-hydroxyhomoserine) or d-glycolaldehyde. This result is consistent with a model in which 4-phospho-hydroxy-l-threonine (synonym, 3-hydroxyhomoserine phosphate), rather than 4-hydroxy-l-threonine, is an obligatory intermediate in pyridoxal 5'-phosphate biosynthesis. Ring closure using 4-phospho-hydroxy-l-threonine as a substrate would lead to the formation of pyridoxine 5'-phosphate, and not pyridioxine, as the first B₆-vitamer synthesized de novo. These considerations suggest that E. coli pyridoxal/pyridoxamine/pyridoxine kinase is not required for the main de novo pathway of pyridoxal 5'-phosphate biosynthesis, and instead plays a role only in the B₆-vitamer salvage pathway.     

The pyridoxal 5' -phosphate site in rabbit skeletal muscle glycogen phosphorylase b: an ultraviolet and 1H and 31P nuclear magnetic resonance spectroscopic study.

1 H NMR spectra of the 3-0-methylpyridoxal 5'-phosphate-n-butylamine reaction product indicated that this analogue forms a Schiff base in aprotic solvent. The uv spectral properties of 3-0-methylpyridoxal-5'-phosphate phosphorylase b correspond to those of the n-butylamine Schiff base derivative in dimethyl sulfoxide. On the basis of that and auxiliary uv and 1H NMR spectra of pyridoxal and pyridoxal 5'-phosphate and the corresponding Schiff base derivatives we have verified that pyridoxal 5' -phosphate is also bound as a Schiff base to phosphorylase and not as an aldamine. Since 3-0-methylpyridoxal-5'-phosphate phosphorylase is active, a proton shuttle between the 3-hydroxyl group and the pyridine nitrogen is excluded. This directs attention to the 5' -phosphate group of the cofactor as a candidate for a catalytic function. 31P NMR spectra of pyridoxal 5' -phosphate in phosphorylase b indicated that deprotonation of the 5' -phosphate group was unresponsive to external pH. Interaction of phosphorylase b with adenosine 5' -monophosphate, the allosteric effector required activity, and arsenate, which substitutes for phosphate as substrate, triggered a conformational change which resulted in deprotonation of the 5' -phosphate group of pyridoxal 5' at pH 7.6. It now behaved like in the pyridoxal-phosphate-epsilon-aminocaproate Schiff base in aqueous buffer, where the diionized form is dominant at this pH. Differences of line widths of the adenosine 5' -monophosphate signal point to different life times of the allosteric effector- enzyme complexes in the presence and absence of substrate (arsenate).     

Evidence for an essential lysine in glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

1. Pyridoxal 5'-phosphate inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides reversibly which Ki equals 0.04-0.06 mM. 2. This inhibition is competitive with respect to glucose 6-phosphate and non-competitive with respect to NADP+ or NAD+. Interaction between enzyme and excess pyridoxal 5'-phosphate follows pseudo-first-order kinetics and indicates that one molecule of inhibitor reacts with each active unit of enzyme. 3. Substrate and coenzyme protect the enzyme from inhibition by pyridoxal 5'-phosphate. Dissociation constants for NADP+ and glucose 6-phosphate were determined from their effects on the kinetics of enzyme--inhibitor interaction. 4. Reaction of the enzyme with pyridoxal 5'-phosphate produces a typical Schiff-base absorbance peak at 430 nm. Subsequent reduction with sodium borohydride leads to spectral changes characteristic for the formation of a secondary amine. 5. The irreversibly inactivated enzyme thus produced contains two moles of inhibitor per mole of enzyme (two subunits per mole). After protein hydrolysis, N-6-pyridoxyllysine can be identified by paper chromatography. 6. The enzyme is inhibited irreversibly by 1-fluoro-2,4-dinitrobenzene, even in the presence of excess 2-mercaptoethanol. At least one dinitrophenyl group is bound per active unit of enzyme; 4 to 5 moles of dinitrophenyl group are bound per mole of enzyme. NADP+ AND GLUCOSE 6-PHOSPHATE PROTECT AGAINST INHIBITION BY 1-FLUORO-2,4-DINITROBENZENE. The absorption spectrum of dinitrophenyl-enzyme corresponds to that for dinitrophenylated amino groups. 7. These studies indicate that there is an essential lysine at the active site of the enzyme. It is suggested that the function of this lysine is to bind glucose 6-phosphate. 8. It is proposed that a group of "active lysine" proteins may exist (in analogy with the "active serine" enzymes), which share a common structural feature at their substrate-binding site and to which pyridoxal 5'-phosphate binds specifically.     

Human brain pyridoxal-5'-phosphate phosphatase (PLPP):protein transduction of PEP-1-PLPP into PC12 cells

Pyridoxal-5'-phosphate phosphatase (PLPP) catalyzes the dephosphorylation of pyridoxal-5'-phosphate (PLP). A human brain PLPP gene was fused with a PEP-1 peptide and produced a genetic in-frame PEP-1-PLPP fusion protein. The purified PEP-1-PLPP fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced PEP-1-PLPP fusion protein was stable for 36 h. The concentration of PLP was markedly decreased by the addition of exogenous PEP-1-PLPP to media pretreated with the vitamin B(6) precursors; pyridoxine, pyridoxal kinase and pyridoxine-5'-phosphate oxidase into cells. The results suggest that the transduction of the PEP-1-PLPP fusion protein can be one mode of PLP level regulation, and to replenish this enzyme in the various neurological disorders related to vitamin B(6).     

Kinetic studies of the pyridoxal kinase from pig liver: slow-binding inhibition by adenosine tetraphosphopyridoxal

Pyridoxal kinase from pig liver has been purified 10,000-fold to apparent homogeneity. The enzyme is a dimer of subunits of Mr 32,000. The enzyme is strongly inhibited by the product pyridoxal 5'-phosphate. Liver pyridoxamine phosphate oxidase, another enzyme involved in the biosynthesis of pyridoxal 5'-phosphate, is also strongly inhibited by this compound [Wada, H., & Snell, E. E. (1961) J. Biol. Chem. 236, 2089-2095]. Thus, the biosynthesis of pyridoxal 5'-phosphate in the liver might be regulated by the product inhibition of both pyridoxamine phosphate oxidase and pyridoxal kinase. Kinetic studies revealed that the catalytic reaction of liver pyridoxal kinase follows an ordered mechanism in which pyridoxal and ATP bind to the enzyme and ADP and pyridoxal 5'-phosphate are released from the enzyme, in this order. Adenosine tetraphosphopyridoxal was found to be a slow-binding inhibitor of pyridoxal kinase. Pre-steady-state kinetics of the inhibition revealed that the inhibitor and the enzyme form an initial weak complex prior to the formation of a tighter and slowly reversing complex. The overall inhibition constant was 2.4 microM. ATP markedly protects the enzyme against time-dependent inhibition by the inhibitor, whereas another substrate pyridoxal affords no protection. By contrast, adenosine triphosphopyridoxal is not a slow-binding inhibitor of this enzyme.     

Purification and characterization of pyridoxine 5'-phosphate phosphatase from Sinorhizobium meliloti

Here we report the purification and biochemical characterization of a pyridoxine 5'-phosphate phosphatase involved in the biosynthesis of pyridoxine in Sinorhizobium meliloti. The phosphatase was localized in the cytoplasm and purified to electrophoretic homogeneity by a combination of EDTA/lysozyme treatment and five chromatography steps. Gel-filtration chromatography with Sephacryl S-200 and SDS/PAGE demonstrated that the protein was a monomer with a molecular size of approximately 29 kDa. The protein required divalent metal ions for pyridoxine 5'-phosphate phosphatase activity, and specifically catalyzed the removal of Pi from pyridoxine and pyridoxal 5'-phosphates at physiological pH (about 7.5). It was inactive on pyridoxamine 5'-phosphate and other physiologically important phosphorylated compounds. The enzyme had the same Michaelis constant (K(m)) of 385 muM for pyridoxine and pyridoxal 5'-phosphates, but its specific constant [maximum velocity (V(max))/K(m)] was nearly 2.5 times higher for the former than for the latter.     

Aminophospholipid glycation and its inhibitor screening system: a new role of pyridoxal 5'-phosphate as the inhibitor

Peroxidized phospholipid-mediated cytotoxity is involved in the pathophysiology of a number of diseases [i.e., the abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) found in the plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (deoxy-D-fructosyl PE, or Amadori-PE), because Amadori-PE causes oxidative stress. However, lipid glycation inhibitor has not been discovered yet because of the lack of a lipid glycation model useful for inhibitor screening. We optimized and developed a lipid glycation model considering various reaction conditions (glucose concentration, temperature, buffer type, and pH) between PE and glucose. Using the developed model, various protein glycation inhibitors (aminoguanidine, pyridoxamine, and carnosine), antioxidants (ascorbic acid, alpha-tocopherol, quercetin, and rutin), and other food compounds (L-lysine, L-cysteine, pyridoxine, pyridoxal, and pyridoxal 5'-phosphate) were evaluated for their antiglycative properties. Pyridoxal 5'-phosphate and pyridoxal (vitamin B(6) derivatives) were the most effective antiglycative compounds. These pyridoxals could easily be condensed with PE before the glucose/PE reaction occurred. Because PE-pyridoxal 5'-phosphate adduct was detectable in human red blood cells and the increased plasma Amadori-PE concentration in streptozotocin-induced diabetic rats was decreased by dietary supplementation of pyridoxal 5'-phosphate, it is likely that pyridoxal 5'-phosphate acts as a lipid glycation inhibitor in vivo, which possibly contributes to diabetes prevention.     

Separation of two phosphorylase kinase phosphatases from rabbit skeletal muscle.

Cyclic-AMP-dependent protein kinase catalyses the activation of phosphorylase kinase and the phosphorylation of two serine residues on the alpha subunit and beta subunit of phosphorylase kinase [Cohen, P., Watson, D.C. and Dixon, G.H. (1975)]. The dephosphorylation of phosphorylase kinase has been shown to be catalysed by two distinct enzymes, termed alpha-phosphorylase kinase phosphatase and beta-phosphorylase kinase phosphatase. These two enzymes show essentially absolute specificity towards the alpha and beta subunits respectively. The two phosphatases copurified through ethanol fractionation, DEAE-cellulose chromatography and ammonium sulphate precipitation, but were separated from each other by a gel filtration on Sephadex G-200. alpha-Phosphorylase kinase phosphatase was purified 500-fold from the ethanol precipitation step, and beta-phosphorylase kinase phosphatase 320-fold. The molecular weights estimated by gel filtration were 170--180 000 for alpha-phosphorylase kinase phosphatase and 75--80 000 for beta-phosphorylase kinase phosphatase. Since the activity of phosphorylase kinase correlates with the state of phosphorylation of the beta subunit (Cohen, P. (1974)), beta-phosphorylase kinase phosphatase is the enzyme which reverses the activation of phosphorylase kinase. alpha-Phosphorylase kinase phosphatase is an enzyme activity that has not been recognised previously. Since the role of the alpha-subunit phosphorylation is to stimulate the rate of dephosphorylation of the beta subunit (Cohen, P. (1974)), alpha-phosphorylase kinase phosphatase can be regarded as the enzyme which inhibits the reversal of the activation of phosphorylase kinase. The implications of these findings for the hormonal control of phosphorylase kinase activity by multisite phosphorylation are discussed.     

Photoaffinity labeling of the glucagon receptor with a new glucagon analog

The flash excitation of the pyridoxal 5'-phosphate cofactor of glycogen phosphorylase b by an ultraviolet laser produces a transient state from a proton transfer of the bound cofactor. The rate of decay of this transient state is sensitive to the ionization state of the cofactor. This proved a useful probe for the ionization state of the 5'-phosphate group of the cofactor on the binding by the enzyme of various substrates. The decay rate data show, for the binding of glucose 1-phosphate, a partially negative 5'-HPO4- and evidence for a PO4-PO4 interaction. The data is interpreted in terms of a dynamic shift of substrates at the active site.     

Pyridoxal phosphate site in glycogen phosphorylase b: structure in native enzyme and in three derivatives with modified cofactors

The detailed environment of the essential cofactor pyridoxal 5'-phosphate in glycogen phosphorylase b, resulting from crystallographic refinement at 1.9-A resolution, is described. The pyridoxal ring is buried in a nonpolar site containing three aromatic rings while the 5'-phosphate group is highly solvated and makes only three direct contacts to the protein. The pyridine nitrogen interacts via a water with protein atoms [main chain carbonyl oxygen (Asn-133) and OH of tyrosine (Tyr-90)]. The crystal structures of three active derivatives of phosphorylase reconstituted with 5'-deoxypyridoxal 5'-methylenephosphonate (PDMP), 6-fluoropyridoxal 5'-phosphate (6-FPLP), and pyridoxal (PL) in place of the natural cofactor have been determined at 2.5-A resolution. The results for PDMP-phosphorylase show a closer proximity of the phosphonate group to the NZ atom of a lysine (Lys-574) than that observed in the native enzyme, consistent with 31P NMR studies that have shown a change in ionization state of the phosphonate group compared to the native cofactor phosphate. The replacement of the polar 5'-ester linkage by a CH2 group results in a small shift of a water and its hydrogen-bonded tyrosine (Tyr-648). In 6-FPLP-phosphorylase the fluorine is accommodated with no significant change in structure. It is suggested that substitution of the electronegative fluorine at the 6-position may result in lower activity of 6-FPLP-phosphorylase through a strengthening of hydrogen-bonded interactions to the pyridine nitrogen N1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae: oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein

Starch phosphorylase from Corynebacterium callunae is a dimeric protein in which each mol of 90 kDa subunit contains 1 mol pyridoxal 5'-phosphate as an active-site cofactor. To determine the mechanism by which phosphate or sulfate ions bring about a greater than 500-fold stabilization against irreversible inactivation at elevated temperatures (> or = 50 degrees C), enzyme/oxyanion interactions and their role during thermal denaturation of phosphorylase have been studied. By binding to a protein site distinguishable from the catalytic site with dissociation constants of Ksulfate = 4.5 mM and Kphosphate approximately 16 mM, dianionic oxyanions induce formation of a more compact structure of phosphorylase, manifested by (a) an increase by about 5% in the relative composition of the alpha-helical secondary structure, (b) reduced 1H/2H exchange, and (c) protection of a cofactor fluorescence against quenching by iodide. Irreversible loss of enzyme activity is triggered by the release into solution of pyridoxal 5'-phosphate, and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display a temperature-dependent degree of melting of secondary structure. By specifically increasing the stability of the dimer structure of phosphorylase (probably due to tightened intersubunit contacts), phosphate, and sulfate, this indirectly (1) preserves a functional active site up to approximately 50 degrees C, and (2) stabilizes the covalent protein cofactor linkage up to approximately 70 degrees C. The effect on thermostability shows a sigmoidal and saturatable dependence on the concentration of phosphate, with an apparent binding constant at 50 degrees C of approximately 25 mM. The extra stability conferred by oxyanion-ligand binding to starch phosphorylase is expressed as a dramatic shift of the entire denaturation pathway to a approximately 20 degrees C higher value on the temperature scale.     

X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with pyridoxal 5'-phosphate at 2.0 A resolution.

Escherichia coli pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate by the FMN oxidation of pyridoxine 5'-phosphate forming FMNH(2) and H(2)O(2). Recent studies have shown that in addition to the active site, pyridoxine 5'-phosphate oxidase contains a non-catalytic site that binds pyridoxal 5'-phosphate tightly. The crystal structure of pyridoxine 5'-phosphate oxidase from E. coli with one or two molecules of pyridoxal 5'-phosphate bound to each monomer has been determined to 2.0 A resolution. One of the pyridoxal 5'-phosphate molecules is clearly bound at the active site with the aldehyde at C4' of pyridoxal 5'-phosphate near N5 of the bound FMN. A protein conformational change has occurred that partially closes the active site. The orientation of the bound pyridoxal 5'-phosphate suggests that the enzyme catalyzes a hydride ion transfer between C4' of pyridoxal 5'-phosphate and N5 of FMN. When the crystals are soaked with excess pyridoxal 5'-phosphate an additional molecule of this cofactor is also bound about 11 A from the active site. A possible tunnel exists between the two sites so that pyridoxal 5'-phosphate formed at the active site may transfer to the non-catalytic site without passing though the solvent.     

Vitamin B(6) salvage enzymes: mechanism, structure and regulation

Vitamin B(6) is a generic term referring to pyridoxine, pyridoxamine, pyridoxal and their related phosphorylated forms. Pyridoxal 5'-phosphate is the catalytically active form of vitamin B(6), and acts as cofactor in more than 140 different enzyme reactions. In animals, pyridoxal 5'-phosphate is recycled from food and from degraded B(6)-enzymes in a "salvage pathway", which essentially involves two ubiquitous enzymes: an ATP-dependent pyridoxal kinase and an FMN-dependent pyridoxine 5'-phosphate oxidase. Once it is made, pyridoxal 5'-phosphate is targeted to the dozens of different apo-B(6) enzymes that are being synthesized in the cell. The mechanism and regulation of the salvage pathway and the mechanism of addition of pyridoxal 5'-phosphate to the apo-B(6)-enzymes are poorly understood and represent a very challenging research field. Pyridoxal kinase and pyridoxine 5'-phosphate oxidase play kinetic roles in regulating the level of pyridoxal 5'-phosphate formation. Deficiency of pyridoxal 5'-phosphate due to inborn defects of these enzymes seems to be involved in several neurological pathologies. In addition, inhibition of pyridoxal kinase activity by several pharmaceutical and natural compounds is known to lead to pyridoxal 5'-phosphate deficiency. Understanding the exact role of vitamin B(6) in these pathologies requires a better knowledge on the metabolism and homeostasis of the vitamin. This article summarizes the current knowledge on structural, kinetic and regulation features of the two enzymes involved in the PLP salvage pathway. We also discuss the proposal that newly formed PLP may be transferred from either enzyme to apo-B(6)-enzymes by direct channeling, an efficient, exclusive, and protected means of delivery of the highly reactive PLP. This new perspective may lead to novel and interesting findings, as well as serve as a model system for the study of macromolecular channeling. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.     

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)     

Interaction of muscle glycogen phosphorylase with pyridoxal 5'-methylenephosphonate

Rabbit muscle glycogen phosphorylase (EC 2.4.1.1) was reconstituted with pyridoxal 5′-methylenephosphonate with ca. 25% restoration of enzymatic activity. The modified enzyme has very similar chemical and physical properties to native phosphorylase including UV and fluorescence spectra, quaternary structure, high energy of activation in the reconstitution reaction, optimum pH and susceptibility to phosphorylase kinase in the b to a conversion. While V_max is reduced to ca. one-fifth, affinities for the substrate glucose 1-P and the effector AMP are increased. This is the first analog of pyridoxal 5′-P modified in the 5′-position found to restore catalytic activity to apophosphorylase.