Metal binding to pyridoxal derivatives. An NMR study of the interaction of Eu(III) with pyridoxal phosphate and pyridoxamine phosphate

The solution conformations of pyridoxal-5′ -phosphate and pyridoxamine-5′-phosphate have been investigated using Eu(III) as a nuclear magnetic resonance shift probe. Binding of Eu(III) to pyridoxal phosphate results in the formation of two complexes, at the phosphate group and theo-hydroxy-aldehyde moiety, which are in slow exchange on the nuclear magnetic resonance time-scale. The lanthanide-induced pseudo contact shifts calculated using the McConnell-Robertson equation (J. Chem. Soc. (1950), 22, 1561) are in good agreement with the experimentally observed values for both pyridoxal phosphate and pyridoxamine phosphate and lead to a family of closely related conformations. Contribution No. 130 from the Molecular Biophysics Unit.     

Observations on the pyridoxal 5'-phosphate inhibition of DNA polymerases.

Pyridoxal 5'-phosphate at concentrations greater than 0.5 mM inhibits polymerization of deoxynucleoside triphosphate catalyzed by a variety of DNA polymerases. The requirement for a phosphate as well as aldehyde moiety of pyridoxal phosphate for inhibition to occur is clearly shown by the fact that neither pyridoxal nor pyridoxamine phosphate are effective inhibitors. Since the addition of nonenzyme protein or increasing the amount of template primer exerted no protective effect, there appears to be specific affinity between pyridoxal phosphate and polymerase protein. The deoxynucleoside triphosphates, however, could reverse the inhibition. The binding of pyridoxal 5'-phosphate to enzyme appears to be mediated through classical Schiff base formation between the pyridoxal phosphate and the free amino group(s) present at the active site of the polymerase protein. Kinetic studies indicate that inhibition by pyridoxal phosphate is competitive with respect to substrate deoxynucleoside triphosphate(s).     

Purification and Properties of 1-AminocycIopropane-l-carboxylate Synthase of Mesocarp of Cucurbita maxima Duch. Fruits

1-Aminocyclopropane-1-carboxylate (ACC) synthase, which formsAGC from S-adenosylmethionine (SAM), was purified to homogeneityfrom sliced and aged mesocarp tissue of Cucurbita maxima Duch.cv Ebisu fruits, and its enzymatic properties were determined.The specific activity of the purified enzyme was 220 mU/mg proteinat 30°C at 50 µM SAM. Native ACC synthase has a relativemolecular mass of 160 ± 10 kDa and consisted of two subunitsof about 84±3 kDa. S-adenosylhomocysteine (SAH), S-methylmethionine(SMM) and L-methionine did not serve as substrate. The enzymereaction was competitively inhibited by aminoethoxyvinylglycine(AVG) (Ki, 2.5 µM), aminooxyacetic acid (Ki, 40 µM)and SAH (Ki, 30 µM). The reaction was also strongly inhibitedby semicarbazide, and less effectively by homocysteine. Theenzyme was rapidly inactivated by its substrate, SAM in thepresence of pyridoxalphosphate (PLP), but in the absence ofPLP, SAM-induced inactivation was much slower. Inactivationdid not occur by SAH and SMM, SAM analogs without substrateactivity. Pyridoxal phosphate was an essential cofactor to beadded to a reaction mixture for maximum activity, but an enzymepreparation from which pyridoxal phosphate was removed by SephadexG-25 gel filtration exhibited one-eighth activity which wasinhibited by semicarbazide, this indicating that a small amountof pyridoxal phosphate is firmly bound to the enzyme. (Received May 6, 1986; Accepted May 20, 1986)     

Modification of Rhodospirillum rubrum ribulose bisphosphate carboxylase with pyridoxal phosphate. 1. Identification of a lysyl residue at the active site.

Ribulose 1,5-bisphosphate carboxylase isolated from Rhodospirillum rubrum was strongly inhibited by low concentrations of pyridoxal 5'-phosphate. Activity was protected by the substrate ribulose bisphosphate and to a lesser extent by other phosphorylated compounds. Pyridoxal phosphate inhibition was enhanced in the presence of magnesium and bicarbonate, but not in the presence of either compound alone. Concomitant with inhibition of enzyme activity, pyridoxal phosphate forms a Schiff base with the enzyme which is reversible upon dialysis and reducible with sodium borohydride. Subsequent to reduction of the Schiff base with tritiated sodium borohydride, tritiated N6-pyridoxyllysine could be identified in the acid hydrolysate of the enzyme. Only small amounts of this compound were present when the reduction was performed in the presence of carboxyribitol bisphosphate, an analogue of the intermediate formed during the carboxylation reaction. Therefore, it is concluded that pyridoxal phosphate modifies a lysyl residue close to or at the active site of ribulose bisphosphate carboxylase.     

Regulation of threonine biosynthesis in barley seedlings (Hordeum vulgare L.)

Homoserine kinase was purified 700-fold by fractional ammonium sulfate precipitation, heat treatment, CM-Sephadex C-50 and DEAE-Sephadex A-50 ion exchange chromatography, and Sephadex G-100 gel filtration. The reaction products O-phosphohomoserine and ADP were the only compounds which caused considerable inhibition of homoserine kinase activity. Product inhibition studies showed non-competitive inhibition between ATP and O-phosphohomoserine and between homoserine and O-phosphohomoserine, and competitive inhibition between ATP and ADP. ADP showed non-competitive inhibition versus homoserine at suboptimal concentrations of ATP. At saturating concentrations of ATP no effect of ADP was observed. The homoserine kinase activity was negligible in the absence of K⁺ and the K_m value for K⁺ was observed to be 4.3 mmol l^–1. A non-competitive pattern was observed with respect to the substrates homoserine and ATP. Threonine synthase in the first green leaf of 6-day-old barley seedlings was partially purified 15-fold by ammonium sulfate fractionation and Sephadex G-100 gel chromatography. Threonine synthase was shown to require pyridoxal 5-phosphate as coenzyme for optimum activity and the enzyme was strongly activated by S-adenosyl-L-methionine. The optimum pH for threonine synthase activity was 7 to 8.Abbreviations PLP Pyridoxal 5-phosphate - SAM S-adenosyl-L-methionine - HSP O-phosphohomoserine     

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.     

Inhibition of the recBC enzyme of Escherichia coli by specific binding of pyridoxal 5'-phosphate to DNA binding site.

Pyridoxal 5'-phosphate rapidly abolished the DNA-hydrolyzing activities as well as DNA-dependent ATP-ase activity of the recBC enzyme of Escherichia coli. Pyridoxal also had an inhibitory effect on the enzyme but less effective than that of pyridoxal 5'-phosphate. Pyridoxamine 5'-phosphate, pyridoxamine, or pyridoxine had no effect on the activities of the enzyme. The inhibition was rapidly reversed by dilution but could be made irreversible by reduction with sodium borohydride prior to dilution. This suggests the formation of Schiff base between pyridoxal 5'-phosphate and an epsilon-amino group of a lysine residue which is essential for the enzyme activity. Pyridoxal 5'-phosphate is a competitive inhibitor of DNA substrate but not of ATP. Furthermore, the presence of DNA substrate protected the enzyme from inactivation by the reduction but the presence of ATP showed no effect. Thus, the recBC enzyme appears to have an essential lysine residue at or near the DNA binding site of the enzyme, and the enzyme possesses two independent catalytic sites, such as a DNA binding site and an ATP binding site.     

Inactivation of the Reconstituted Oxoglutarate Carrier from Bovine Heart Mitochondria by Pyridoxal 5′-Phosphate

The effect of pyridoxal 5-phosphate and some other lysine reagents on the purified,reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition ofoxoglutarate/oxoglutarate exchange by pyridoxal 5-phosphate can be reversed by passing theproteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodiumborohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal5-phosphate, which caused a time- and concentration-dependent inactivation of oxoglutaratetransport with an IC₅₀ of 0.5 mM, competed with the substrate for binding to the oxoglutaratecarrier (K _i = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by pyridoxal5-phosphate indicated that modification of a single amino acid residue/carrier molecule wassufficient for complete inhibition of oxoglutarate transport. After reduction with sodiumborohydride [³H]pyridoxal 5-phosphate bound covalently to the oxoglutarate carrier. Incubation ofthe proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivationand no radioactivity was found associated with the carrier protein. In contrast, glutarate andsubstrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl,which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier againstinhibition by pyridoxal 5-phosphate. These results indicate that pyridoxal 5-phosphateinteracts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminalglycine residue) which is essential for substrate translocation and may be localized at or nearthe substrate-binding site.     

Chemical modification of intracellular cytosine deaminase fromChromobacterium violaceum YK 391

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. Amino acid residues located in or near the active sites of the intracellular cytosine deaminase fromChromobacterium violaceum YK 391 were identified by chemical modification studies. The enzymic activity was completely inhibited by chemical modifiers, such as 1 mM NBS, chloramine-T, ρ-CMB, ρ-HMB and iodine, and was strongly inhibited by 1 mM PMSF and pyridoxal 5′-phosphate. This chemical deactivation of the enzymic activity was reversed by a high concentration of cytosine. Furthermore, the deactivation of the enzymic activity by ρ-CMB was also reversed by 1 mM cysteine-HCl, DTT and 2-mercaptoethanol. These results suggested that cysteine, tryptophan and methionine residues might be located in or near the active sites of the enzyme, while serine and lysine were indirectly involved in the enzymic activity. The intracellular cytosine deaminase fromC. violaceum YK 391 was assumed to be a thiol enzyme.     

Brain pyridoxal kinase. Mobility of the substrate pyridoxal and binding of inhibitors to the nucleotide site.

Pyridoxal kinase has been purified 2000-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. The interactions of the substrate pyridoxal and the inhibitor N-dansyl-2-oxopyrrolidine (dansyl = 5-dimethylaminonaphthalene-1-sulfonyl) with the catalytic site were examined by means of fluorescence spectroscopy. The increase in emission anisotropy that follows the binding of pyridoxal to the kinase was used to determine the equilibrium dissociation constant. Pyridoxal kinase binds one molecule of substrate with a Kd = 11 microns at pH 6. The emission anisotropy spectrum of bound pyridoxal reveals that the substrate is not rigidly trapped by the protein matrix. N-Dansyl-2-oxopyrrolidine is a competitive inhibitor with respect to ATP at saturating concentrations of pyridoxal. It binds to the enzyme with a dissociation constant of 6 microns. N-Dansyl-2-oxopyrrolidine is immobilized by strong interactions with the enzyme, but it is displaced from the catalytic site by ATP. The results are consistent with the hypothesis that N-dansyl-2-oxopyrrolidine binds at the nucleotide binding site of pyridoxal kinase.     

Function of the phosphate group of pyridoxal 5'-phosphate in the glycogen phosphorylase reaction

To understand the catalytic mechanism of glycogen phosphorylase (EC 2.4.1.1), pyridoxal(5')phospho(1)-beta-D-glucose was synthesized and examined as a hypothetical intermediate in the catalysis. Pyridoxal phosphoglucose bound stoichiometrically to the cofactor site of rabbit muscle phosphorylase b in a similar mode of binding to the natural cofactor, pyridoxal 5'-phosphate. The rate of binding of pyridoxal phosphoglucose was only 1/100 compared with that of pyridoxal phosphate. The enzyme reconstituted with pyridoxal phosphoglucose showed no enzymatic activity at all even after prolonged incubation of the enzyme with substrates and activator. The present data would contradict participation of the phosphate group of pyridoxal phosphate in a covalent glucosyl-enzyme intermediate even if the covalent intermediate was formed during the catalysis.     

Purification and properties of diaminopimelate decarboxylase ofMicrococcus glutamicus

Diaminopimelate decarboxylase (EC 4.1.1.20) ofMicrococcus glutamicus ATCC 13059 was purified to homogeneity. The enzyme had an apparent molecular weight of 191,000 as determined by gel filtration on Sephadex G-200. At protein concentrations of 20 and 10 μg per ml and in the absence of pyridoxal-5′-phosphate, it dissociated into a species of molecular weight 94,000. The polypeptide chain molecular weight as determined by sodium dodecyl sulphate Polyacrylamide gel electrophoresis was 100,000. TheK _m formeso diaminopimelate was 0.5 mM and that for pyridoxal-5′-phosphate was 0.6 μI. Sulphydryl groups and pyridoxal-5′-phosphate were essential for activity and stability. The enzyme was inhibited significantly by L-lysine and DL-aspartic β-semialdehyde.     

Proposals for the catalytic mechanism of glycogen phosphorylase b prompted by crystallographic studies on glucose 1-phosphate binding

The crystal structure of glycogen phosphorylase b in the presence of the weak activator 2 mm-inosine 5′-phosphate has been solved at 3 Å resolution. The binding interactions of the substrate, glucose 1-phosphate, at the catalytic site are described. The nearby presence (6 Å) of the essential co-factor, pyridoxal phosphate, is consistent with biochemical studies but an analysis of the way in which this group might act in catalysis leads to results that are inconsistent with solution studies. Moreover it is difficult to accommodate a glycogen substrate with its terminal glucose in the position defined by glucose 1-phosphate. Model-building studies show that an alternative binding mode for glucose 1-phosphate is possible and that this alternative mode allows a glycogen substrate to be fitted with ease. The alternative binding site leads directly to proposals for the mechanism in which the phosphate group of pyridoxal phosphate acts as a nucleophile and the imidazole of histidine 376 functions as a general acid. It is suggested that these are the essential features of the catalytic mechanism and that, in the absence of the second substrate, glycogen, and in the absence of AMP, the enzyme binds glucose 1-phosphate in a non-productive mode. Conversion of the enzyme to the active conformation through association with AMP may result in conformational changes that direct the binding to the productive mode.     

Active-site modification of glycerol-3-phosphate dehydrogenase by pyridoxal-5′-phosphate

Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from rabbit skeletal muscle is inhibited by pyridoxal-5′-phosphate. The inhibition observed in steady-state kinetic studies is competitive with respect to dihydroxyacetone phosphate and uncompetitive with respect to NADH. Similar inhibition was found for a series of related compounds which in order of increasing effectiveness of inhibition were: 4-deoxypyridoxine < pyridoxal < pyridoxic acid < pyridoxal-5′-phosphate < pyridoxine and pyridoxamine-5′-phosphate. Pyridoxal-5′-phosphate also reacts slowly with the enzyme to produce an adduct which upon treatment with sodium borohydride results in irreversible modification of the enzyme. The nature of the adduct was investigated by titration of the enzyme with pyridoxal-5′-phosphate, uv-visible and fluorescence spectroscopy, amino acid analysis, and peptide mapping. All such studies are consistent with a single, highly reactive lysyl residue on each enzyme subunit. Protection of the lysyl residue against modification was afforded by the presence of NADH. The modified enzyme, on the other hand, possessed kinetic properties similar to the native enzyme including a nearly identical inhibition constant for pyridoxal-5′-phosphate. Pyridoxal-5′-phosphate, therefore, seems to have two sites of interaction on the enzyme: a reversible binding site competitive with substrate and a Schiff-base site protected by NADH. These properties of glycerol-3-phosphate dehydrogenase set it apart from functionally similar enzymes.     

Kinetic and structural studies of the role of the active site residue Asp235 of human pyridoxal kinase

Pyridoxal kinase catalyzes the phosphorylation of pyridoxal (PL) to pyridoxal 5′-phosphate (PLP). A D235A variant shows 7-fold and 15-fold decreases in substrate affinity and activity, respectively. A D235N variant shows ∼2-fold decrease in both PL affinity and activity. The crystal structure of D235A (2.5 Å) shows bound ATP, PL and PLP, while D235N (2.3 Å) shows bound ATP and sulfate. These results document the role of Asp235 in PL kinase activity. The observation that the active site of PL kinase can accommodate both ATP and PLP suggests that formation of a ternary Enz·PLP·ATP complex could occur in the wild-type enzyme, consistent with severe MgATP substrate inhibition of PL kinase in the presence of PLP.     

Destabilization of tyrosine aminotransferase by amino acids

Summary Several L-amino acids (tyrosine, glutamate, methionine, tryptophan, and phenylalanine) and penicillamine destabilized purified tyrosine aminotransferase by removing enzyme-bound pyridoxal 5-phosphate. The destabilization was measured as a progressive loss of enzyme activity in samples taken at intervals from a primary mixture that was incubated at 37°C. Each destabilizing amino acid either served as a substrate for this enzyme or was a product of transamination. In contrast, L-cysteine destabilized the enzyme only if liver homogenate was added, which generated polysulfide by desulfuration. Cysteine complexed free pyridoxal-5-phosphate but did not remove it from the enzyme. Other amino acids did not destabilize tyrosine aminotransferase at the concentrations tested.Abbreviations TyrAT tyrosine aminotransferase (E.C. 2.6.1.5) - PLP pyridoxal-5-phosphate     

Effect of tryptophan metabolites on the activities of rat liver pyridoxal kinase and pyridoxamine 5-phosphate oxidase in vitro.

Pyridoxal kinase was purified 4760-fold from rat liver. The Km values for pyridoxine and pyridoxal were 120 and 190 microM respectively, and pyridoxine showed substrate inhibition at above 200 microM. Pyridoxamine 5-phosphate oxidase was also purified 2030-fold from rat liver, and its Km values for pyridoxine 5-phosphate and pyridoxamine 5-phosphate were 0.92 and 1.0 microM respectively. Pyridoxine 5-phosphate gave a maximum velocity that was 5.6-fold greater than with pyridoxamine 5-phosphate and showed strong substrate inhibition at above 6 microM. Among the tryptophan metabolites, picolinate, xanthurenate, quinolinate, tryptamine and 5-hydroxytryptamine inhibited pyridoxal kinase. However, pyridoxamine 5-phosphate oxidase could not be inhibited by tryptophan metabolites, and on the contrary it was activated by 3-hydroxykynurenine and 3-hydroxyanthranilate. Regarding the metabolism of vitamin B-6 in the liver, the effects of tryptophan metabolites that were accumulated in vitamin B-6-deficient rats after tryptophan injection were discussed.     

Studies on determination of active site amino acid residues in glyoxylate synthetase from potato tuber chloroplasts

A homogeneous preparation of glyoxylate synthetase from greening potato tubers was used to study the functional role of disulphide groups, lysine and tryptophan residues in enzyme catalysis. The formation of a thioisoindole derivative was demonstrated by spectral analysis of the reduced and o-phthalaldehyde-treated enzymes. o-Phthalaldehyde modification resulted in about a 25 % loss of tryptophan emission at 336 nm and the appearance of a 410-nm emission peak characteristic of a thioisoindole. Ferrous iron was capable of generating thiol groups and addition of substrate resulted in a faster disappearance of these thiols. The optimal time for maximum glyoxylate synthesis by glyoxylate synthetase paralleled the disappearance of these thiols. Involvement of lysine and tryptophan residues in the enzyme reaction was demonstrated by the inhibition of activity by pyridoxal 5′-phosphate and dimethyl(2-hydroxy 5-nitrobenzyl) sulphonium bromide (DMHNB), respectively. Pyridoxal phosphate strongly and reversibly inhibited glyoxylate synthetase, and substrate and metal ion provided significant protection against inhibition. The results suggest that the lysine residue may be at or near the active binding site. The lysyl residue formed a Schiff base with pyridoxal phosphate which was stabilised by NaBH₄. Glyoxylate synthetase was also irreversibly inactivated by a tryptophan selective reagent, DMHNB, while substrate provided substantial protection against inactivation. Kinetic analysis and correlation of the spectral data at 410 nm indicated that complete inactivation by DMHNB resulted from the modification of 5 tryptophan residues/subunit, of which one was essential for activity. The available evidence suggests a possible concerted action of enzyme disulphides, ferrous iron, lysine and aromatic amino acid residues in the synthesis of glyoxylate by this enzyme.     

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.     

A histidine decarboxylase-like mRNA is involved in tomato fruit ripening

DNA sequencing of a tomato ripening-related cDNA, TOM 92, revealed an open reading frame with homology to several pyridoxal 5-phosphate histidine decarboxylases, containing the conserved amino acid residues known to bind pyridoxal phosphate and -fluoromethylhistidine, an inhibitor of enzyme activity. TOM 92 mRNA accumulated during early fruit ripening and then declined. Fruit of the ripeningimpaired tomato mutant, ripening inhibitor (rin), did not accumulate TOM 92 mRNA, and its accumulation was not restored by treatment of fruit with ethylene. The TOM 92 mRNA was not detected in tomato leaves and unripe fruit.