The effect of pyridoxal phosphate on the activity of aldolase from Lemna minor L.

Lemna aldolase has been purified by ion-exchange and affinity chromatography. The enzyme is inhibited by pyridoxal phosphate in a manner which suggests that pyridoxal phosphate forms a non-covalent complex with the enzymes which is in equilibrium with the Schiff base covalently modified enzyme. The kinetics of the reversal of inhibition have been used to test the proposition that the fall in aldolase activity observed during periods of nitrogen starvation is due to inhibition by pyridoxal phosphate. It is concluded that the in vivo loss of aldolase activity is not due to pyridoxal phosphate and that the in vitro inhibition of glycolytic enzymes by pyridoxal phosphate is due to the reaction with lysine residues at the active sites which are necessary to bind the strongly acidic sugar phosphates.     

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.     

Arginine decarboxylase from Lathyrus sativus seedlings. Purification and properites.

Arginine decarboxylase which makes its appearance in Lathyrus sativus seedlings after 24 h of seed germination reaches its highest level around 5-7 days, the cotyledons containing about 60% of the total activity in the seedlings at day 5. The cytosol enzyme was purified 977-fold from whole seedlings by steps involving manganese chloride treatment, ammonium sulphate and acetone fractionations, positive adsorption on alumina C-gamma gel, DEAE-Sephadex chromatography followed by preparative disc gel electrophoresis. The enzyme was shown to be homogeneous by electrophoretic and immunological criteria, had a molecular weight of 220,000 and appears to be a hexamer with identical subunits. The optimal pH and temperature for the enzyme activity were 8.5 and 45 degrees C respectively. The enzyme follows typical Michaelis-Menten kinetics with a Km value of 1.73 mM for arginine. Though Mn2+ at lower concentrations stimulated the enzyme activity, there was no dependence of the enzyme on any metal for the activity. The arginine decarboxylase of L. sativus is a sulfhydryl enzyme. The data on co-factor requirement, inhibition by carbonyl reagents, reducing agents and pyridoxal phosphate inhibitors, and a partial reversal by pyridoxal phosphate of inhibition by pyridoxal-HCl suggests that pyridoxal 5'-phosphate is involved as a co-factor for the enzyme. The enzyme activity was inhibited competitively by various amines including the product agmatine. Highest inhibition was obtained with spermine and arcain. The substrate analogue, L-canavanine, homologue L-homoarginine and other basic amino acids like L-lysine and L-ornithine inhibited the enzyme activity competitively, homoarginine being the most effective in this respect.     

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).     

Location of the reactive sulfhydryl residues in the primary sequence of the β2 subunit of tryptophan synthase of Escherichiacoli

Two of the 5 sulfhydryl residues of the β₂ subunit of tryptophan synthase have previously been shown to react with N-ethylmaleimide and to have active site roles. We now show that the single sulfhydryl which reacts with N-ethylmaleimide in the presence of pyridoxal phosphate is cysteine-170. The essential sulfhydryl which reacts with N-ethylmaleimide or with 2-nitro-5-thiocyanobenzoic acid after removal of pyridoxal phosphate is cysteine-230. The affinity reagent, bromoacetylpyridoxamine phosphate, reacts variably with cysteine-62 or with cysteine-230.     

Studies of the function and location of two cysteines in the beta 2 subunit of tryptophan synthase.

Our findings that the apo β₂ subunit of tryptophan synthase of Escherichia coli is inactivated by the modification of one sulfhydryl residue per monomer by nitrothiocyanobenzoic acid and is reactivated by removal of the CN group indicate that the reactive sulfhydryl residue (SH-I) is essential for catalytic activity. SH-I is shown to be the same residue which was previously found to react with bromoacetylpyridoxamine phosphate and different from a sulfhydryl (SH-II) which reacts with N-ethylmaleimide in the presence of pyridoxal phosphate. The results of partial tryptic digestions of β₂ subunit labeled selectively at SH-I or SH-II show that both sulfhydryl residues are located in the F₁ fragment which also contains the pyridoxal phosphate binding site.     

Chemical modification in situ of Escherichia coli 30 S ribosomal proteins by the site-specific reagent pyridoxal phosphate. Inactivation of the aminoacyl-tRNA and mRNA binding sites

epsilon-Amino groups of lysines of 30 S ribosomal subunits with affinity for phosphate groups were selectively modified in situ by reaction with pyridoxal phosphate and reduction of the Schiff base with nonradioactive or radioactive sodium borohydride. This reaction modified only a limited number of ribosomal proteins and resulted in the loss of only some 30 S activities. The modified proteins were identified and the extent of their modification determined. The main targets of the reaction were S3 greater than S1 greater than S6. The activity most severely affected by the pyridoxal phosphate reaction was mRNA-dependent aminoacyl-tRNA binding. Some inhibition of poly(U) binding was also observed, while neither binding of initiation factors nor association with 50 S subunits was inhibited. The inhibition of aminoacyl-tRNA binding showed distinct selectivity: the inhibition was far greater with NAcPhe-tRNA than with fMet-tRNA and with "A" site than with "P" site binding. In addition, initiation complex formation with some mRNAs (e.g. MS2 RNA) was affected more than with others (e.g. T7 early mRNA). Ribosome reconstitution experiments showed that the modification of protein S3 was the primary cause of the inhibition; a role was also played by ribosomal proteins S1, S2, and S21. Substrate protection experiments showed that the 30 S activity can be protected from pyridoxal phosphate inactivation upon formation of a ternary complex with poly(U) and tRNAPhe or NAcPhe-tRNAPhe. Accordingly, the extent of modification of ribosomal protein S3 was reduced in the ternary complex while modification of S1 was reduced in the presence of poly(U) alone.     

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.     

The inhibition of lysyl oxidase in vivo by isoniazid and its reversal by pyridoxal. Effect on collagen cross-linking in the chick embryo.

Isonicotinic acid hydrazide (isoniazid) causes a large increase in the salt-solubility of collagen when injected into chick embryos; this change is accompanied by the inactivation of lysyl oxidase (EC 1.4.3.13), the enzyme responsible for initiating cross-link formation in collagen and elastin. In addition, isoniazid markedly decreases the liver content of pyridoxal phosphate. The depletion of pyridoxal phosphate takes approx. 6 h, whereas the inhibition of lysyl oxidase and the increase in collagen solubility occur more slowly. A reversal of these effects of isoniazid can be produced by the subsequent injection of a stoichiometric amount of pyridoxal, supporting the role of pyridoxal as a cofactor for lysyl oxidase. Treatment of chick embryos with beta-aminopropionitrile, an irreversible inhibitor of lysyl oxidase, causes an inhibition of the enzyme, which begins to recover within 24 h but which is not affected by the administration of pyridoxal; with isoniazid inhibition, however, lysyl oxidase activity does not show any sign of recovery by 48 h. It is proposed that isoniazid may cause the inhibition of lysyl oxidase by competing for its obligatory cofactor, pyridoxal phosphate. The potential clinical implications in the therapeutic control of fibrosis are briefly discussed.     

Structure-based development of pyridoxal propionate derivatives as specific inhibitors of cathepsin K in vitro and in vivo

We found that pyridoxal phosphate shows considerable inhibition of cathepsins. CLIK-071, in which the phosphate ester of position 3 of pyridoxal phosphate was replaced by propionate, strongly inhibited cathepsin B. Three new types of synthetic pyridoxal propionate derivatives showing specific inhibition of cathepsin K were developed. New synthetic pyridoxal propionate derivatives, -162, -163, and -164, in which the methyl arm of position 6 of CLIK-071 was additionally modified, strongly inhibited cathepsin K and cathepsin S weakly, but other cathepsins were not inhibited. CLIK-166, in which the position 4 aldehyde of CLIK-071 is replaced by a vinyl radical and position 5 is additionally modified, showed cathepsin K-specific inhibition at 10(-5) M. Pit formation due to bone collagen degradation by cathepsin K of rat osteoclasts was specifically suppressed by administration of CLIK-164, but not by inhibitors of cathepsin L or B.     

Reversible modification of amino groups in aspartate aminotransferase.

Amino groups in the pyridoxal phosphate, pyridoxamine phosphate, and apo forms of pig heart cytoplasmic aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC .2.6.1.1) have been reversibly modified with 2,4-pentanedione. The rate of modification has been measured spectrophotometrically by observing the formation of the enamine produced and this rate has been compared with the rate of loss of catalytic activity for all three forms of the enzyme. Of the 21 amino groups per 46 500 molecular weight, approx. 16 can be modified in the pyridoxal phosphate form with less than a 50% change in the catalytic activity of the enzyme. A slow inactivation occurs which is probably due to reaction of 2,4-pentanedione with the enzyme-bound pyridoxal phosphate. The pyridoxamine phosphate enzyme is completely inactivated by reaction with 2,4-pentanedione. The inactivation of the pyridoxamine phosphate enzyme is not inhibited by substrate analogs. A single lysine residue in the apoenzyme reacts approx. 100 times faster with 2,4-pentanedione than do other amino groups. This lysine is believed to be lysine-258, which forms a Schiff base with pyridoxal phosphate in the holoenzyme.     

Chemical and kinetic mechanisms of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli.

The chemical and kinetic mechanisms of purified aspartate-beta-semialdehyde dehydrogenase from Escherichia coli have been determined. The kinetic mechanism of the enzyme, determined from initial velocity, product and dead end inhibition studies, is a random preferred order sequential mechanism. For the reaction examined in the phosphorylating direction L-aspartate-beta-semialdehyde binds preferentially to the E-NADP-Pi complex, and there is random release of the products L-beta-aspartyl phosphate and NADPH. Substrate inhibition is displayed by both Pi and NADP. Inhibition patterns versus the other substrates suggest that Pi inhibits by binding to the phosphate subsite in the NADP binding site, and the substrate inhibition by NADP results from the formation of a dead end E-beta-aspartyl phosphate-NADP complex. The chemical mechanism of the enzyme has been examined by pH profile and chemical modification studies. The proposed mechanism involves the attack of an active site cysteine sulfhydryl on the carbonyl carbon of aspartate-beta-semialdehyde, with general acid assistance by an enzyme lysine amino group. The resulting thiohemiacetal is oxidized by NADP to a thioester, with subsequent attack by the dianion of enzyme bound phosphate. The collapse of the resulting tetrahedral intermediate leads to the acyl-phosphate product and liberation of the active site cysteine.     

Chemical modification of the lysine residues of bacterial formate dehydrogenase

Inactivation of formate dehydrogenase by formaldehyde, pyridoxal and pyridoxal phosphate was studied. The effects of concentrations of the modifying agents, substrates, products and inhibitors on the extent of the enzyme inactivation were examined. A complete formate dehydrogenase inactivation by pyridoxal, pyridoxal, phosphate and formaldehyde is achieved by the blocking of 2, 5 and 13 lysine residues per enzyme subunit, respectively. The coenzymes do not protect formate dehydrogenase against inactivation. In the case of modification by pyridoxal and pyridoxal phosphate a complete maintenance of the enzyme activity and specific protection of one lysine residue per enzyme subunit is observed during formation of a binary formate-enzyme complex, or a ternary enzyme--NAD--azide complex. One lysine residue is supposed to be located at the formate-binding site of the formate dehydrogenase active center.     

Reactivation and reconstitution of glutamate decarboxylase upon the interaction of its dimers with pyridoxal phosphate]

The relationship between the reactivation and reconstitution of the hexameric form of glutamate decarboxylase during the interaction of inactive apoenzyme dimers with pyridoxal phosphate (PLP) has been studied. It was shown that the restoration of enzymatic activity, appearance of spectral maximum at 340 nm, and reconstitution of the hexamer depend on the amount of PLP added; this reaction is completed when the PLP concentration reaches that of the initial enzyme. This native hexamer of the holo- and apoenzyme does not practically contain exposed sulfhydryl groups. Ten cysteine residues become available after DS-Na denaturation. The dimer of the apoenzyme contains 8 exposed and 2 buried cysteine residues. The hexamer formation from the dimers is accompanied by the burying of the cysteine residues. When half of the required PLP was added, 7 cysteine residues became buried in experiments with DTNB and six in experiments with 4.4'-DTDP. Further addition of PLP led to the disappearance of the exposed sulfhydryl groups.     

Evidence for the regulation of pyridoxal 5′-phosphate formation in liver by pyridoxamine (pyridoxine) 5′-phosphate oxidase

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5′-phosphate. The K_i, 3 μM, is considerably lower than the K_m for either natural substrate (18 and 24 μM for pyridoxamine 5′-phosphate and 25 and 16 μM for pyridoxine 5′-phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The K_i determined using a 10% rabbit liver homogenate is the same as that for the pure enzyme; hence, product inhibition $\text{in}\text{vivo}$ is probably not diminished significantly by other cellular components. Similar determinations for a 10% rat liver homogenate also show strong inhibition by pyridoxal 5′-phosphate. Since the reported liver content of free or loosely bound pyridoxal 5′-phosphate is greater than K_i, the oxidase in liver is probably associated with pyridoxal 5′-phosphate. These results also suggest that product inhibition of pyridoxamine-P oxidase may regulate the $\text{in}\text{vivo}$ rate of pyridoxal 5′-phosphate formation.     

Inhibitors of crayfish glutamic acid decarboxylase

Crayfish glutamic acid decarboxylase (GAD), like the homologous enzymes from other species, is inhibited by carbonyl-trapping agents (e.g. aminooxyacetic acid; AOAA) and sulfhydryl reagents (e.g. 5,5-dithiobis-(2-nitrobenzoic acid); DTNB). It also is inhibited by the product GABA, many anions (e.g. SCN^– and Cl^–), and some cations (e.g. Zn⁺²). The inhibition by AOAA, but not that by DTNB, was prevented by increasing the concentration of the pyridoxal phosphate (PLP) coenzyme. GABA blocked the effects of PLP on enzyme activity. The inhibition by AOAA, DTNB, GABA, and chloride all were competitive with substrate. The effect of GABA occurs at physiological concentrations and may contribute to the regulation of GAD activity in vivo. The quantitative effect of anions is dependent on the cation with which they are administered. ATP stimulated GAD activity in homogenates prepared with potassium phosphate or Tris-acetate buffer, even when no exogenous PLP was provided.     

The control of plant glutamate dehydrogenase by pyridoxal-5′-phosphate

The proposition that the nitrogen status of a plant is reflected by the ratio pyridoxal phosphate to pyridoxamine phosphate and that this ratio exerts a controlling influence on plant metabolism has been examined. The ratio pyridoxal phosphate to pyridoxamine phosphate has been shown to increase during nitrogen starvation. The inhibition of glutamate dehydrogenase by pyridoxal phosphate has been examined and the kinetics of inhibition are discussed in relation to the proposed control of metabolism.     

Mechanism of inhibition of thrombin-induced platelet aggregation by pyridoxal phosphate

Thrombin and ADP-induced platelet aggregation are reversibly inhibited by pyridoxal phosphate. Sodium borohydride converts Schiff bases formed between pyridoxal phosphate and amino groups to covalent bonds. When platelets treated with sodium borohydride and pyridoxal phosphate are resuspended in fresh platelet-poor plasma, they recover their response to thrombin, but not to ADP. Thus Schiff base formation between pyridoxal phosphate and platelet surface amino groups does not block thrombin aggregation. The loss of thrombin potency as an aggregating agent is due to interaction between pyridoxal phosphate and thrombin. This is evidenced by spectrophometric determination of adduct formation and loss of hydrolytic action on p-tosyl-L-arginine methyl ester.     

Mitochondrial nicotinamide nucleotide transhydrogenase: inhibition by ethoxyformic anhydride, dansyl chloride, and pyridoxal phosphate

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH; EC 1.6.1.1) was inactivated by treatment with pyridoxal phosphate, ethoxyformic anhydride (EFA) or dansyl chloride. NADP and NADPH, but not NAD and NADH, protected TH against inhibition by pyridoxal phosphate, and L-lysine reversed this inhibition. The results suggested modification of an essential lysyl residue by pyridoxal phosphate, possibly at the NADP(H) binding site of TH. EFA and dansyl chloride inhibited TH in a similar manner. The effect of pH on the rate of inhibition of TH by EFA and dansyl chloride was the same, and in both cases addition of NADP and particularly NADPH accelerated the rate of inhibition, while addition of NAD or NADH had no effect. Double inhibition studies, using in one experiment dithiothreitol-reversible inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) to protect the thiol groups of TH, and in another experiment lysine-reversible inhibition by pyridoxal phosphate to protect the putative essential lysyl residues of the enzyme, followed in each case by further treatment of the protected TH with EFA or dansyl chloride, suggested that the inhibitions by EFA and dansyl chloride were independent of the inhibitions by 5,5'-dithiobis (2-nitrobenzoic acid) and pyridoxal phosphate. The inhibitors discussed above are interesting, because pyridoxal phosphate is the only reagent known which appears to modify an essential residue in the NADP(H), but not the NAD(H), binding site of TH, and EFA and dansyl chloride are the only inhibitors known which appear to react with essential residues outside the active site of TH. It is possible that EFA and dansyl chloride inhibitions involve modification of essential prototropic residues in the proton translocation domain of the enzyme.     

Kinetic and thermodynamic parameters for Schiff base formation of vitamin B6 derivatives with amino acids

Thermodynamic and kinetic parameters for Schiff base formation of pyridoxal 5'-phosphate and pyridoxal with epsilon-aminocaproic acid as well as of pyridoxal 5'-phosphate with L-serine were obtained in 0.1 M sodium pyrophosphate buffer as a function of temperature. Changes in enthalpy, which were determined by direct microcalorimetry, were small at 25 degrees C, but varied strongly with pH for the reaction of pyridoxal 5'-phosphate with the amino acids. In contrast to the fast Schiff base formation of pyridoxal 5'-phosphate, a very slow reaction was found for pyridoxal and epsilon-aminocaproic acid concomitant with a larger change in enthalpy. By preventing hemiacetal formation the phosphate moiety plays a crucial role.