Pyridoxamine-5'-phosphate oxidase exhibits no specificity in prochiral hydrogen abstraction from substrate

The stereochemistry for hydrogen removal from pyridoxamine 5'-phosphate with liver pyridoxine (pyridoxamine)-5'-phosphate oxidase was examined to determine whether or not there are significant steric constraints at the substrate region of the active site of the oxidase. For this, pyridoxal 5'-phosphate was reduced with tritium-labeled sodium borohydride in ammoniacal solution to yield racemically labeled [4',4'-3H]pyridoxamine 5'-phosphate which was then chemically or enzymatically oxidized to [4'-3H]pyridoxal 5'-phosphate. This latter was used as coenzyme with either L-aspartate (L-glutamate) aminotransferase and L-glutamate or L-glutamate decarboxylase and alpha-methyl-DL-glutamate to generate [4'-3H]pyridoxamine 5'-phosphate known to be labeled in the R-position. Reaction of the oxidase with the pro-R as well as the pro-R,S-labeled substrates followed by isolation of [4'-3H]pyridoxal 5'-phosphate and 3H2O revealed only half the radioactivity was abstracted from the original substrate in either case. Hence, the oxidase is not stereospecific and equally well catalyzes removal of either pro-R or pro-S hydrogen from the 4-methylene of pyridoxamine 5'-phosphate.     

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.     

Determination of vitamin B6 vitamers and pyridoxic acid in plasma: development and evaluation of a high-performance liquid chromatographic assay

Marginal deficiency of vitamin B6 has recently been related to cardiovascular diseases. Because of that there is an increasing interest in a suitable and reliable method for quantifying this vitamin in routine laboratory medicine. We have developed a HPLC-based method able to quantify the B6 vitamers pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxamine 5'-phosphate (PMP), pyridoxine (PN), and pyridoxamine (PM) and the degradation product 4-pyridoxic acid (4-PA). The separation was accomplished using a C18 (ODS) analytical column and an ion-pair reversed-phase chromatography. B6 vitamers were eluted with a gradient of acetonitrile (0.5-15%) in a potassium phosphate buffer with 1-octanesulfonic acid and triethylamine, pH 2.16. The concentration of the vitamers was determined with fluorescence detector (328 nm excitation, 393 nm emission) after postcolumn derivatization with phosphate buffer containing 1 g/L sodium bisulfite. The performance of the assay was evaluated by analyzing six plasma samples with interrelated concentration and two control samples (unspiked and vitamer spiked) over a 3-months period. The HPLC method was able to identify PLP, 4-PA, PM, PL, PN, and PMP from all other compounds in plasma in an analytical run of 46 min. The imprecisions and mean values (presented in parenthesis in nmol/L) were (unspiked and spiked sample) 9-8% (41-65) for PLP, 12-7% (18-40) for 4-PA, 67-28% (4-19) for PL, 15% (21) for PN, 10% (27) for PM, and 27% (17) for PMP. All three B6 vitamers (PLP, 4-PA, and PL) present in unspiked plasma showed an excellent linearity within the range of (nM) 8-60 (4-PA), 1-19 (PL), and 11-99 (PLP). In conclusion, we report a HPLC-based method that separates and detects nanomolar quantities of six B6 vitamers and demonstrate that the method will be suitable for routine quantitation of PLP and 4-PA in human plasma.     

EFFECT OF HYPERPHENYLALANINEMIA ON VITAMIN B6, METABOLISM IN DEVELOPING RAT BRAIN

Abstract— The turnover of the different forms of B₆ vitamers in the brains of normal and hyperphenylalaninemic preweanling rats was compared after administration of a load of [¹⁴C]pyridoxol. Metabolic transformations occurred in the following sequence: oxidation of pyridoxol to pyridoxal, which was in turn phosphorylated to the 5'-phosphate ester. No significant amount of pyridoxamine was formed during the 8-h experimental period. Pyridoxamine 5'-phosphate was derived from pyridoxal 5'-phosphate. The specific radioactivity of pyridoxal phosphate in the hyperphenylalaninemic brain was significantly lower and increased at a slower rate than in control brains. This difference could not be accounted for by either a deficient supply or inhibited activity of the enzyme, pyridoxal kinase. The synthesis of pyridoxamine 5'-phosphate in the experimental animals also lagged behind the controls. Decreased activity of enzymes dependent on pyridoxal phosphate as cofactor would explain the slower turnover of this B₆-coenzyme.     

Expression, purification, and kinetic constants for human and Escherichia coli pyridoxal kinases

Pyridoxal kinase is an ATP dependent enzyme that phosphorylates pyridoxal, pyridoxine, and pyridoxamine forming their respective 5'-phosphorylated esters. The kinase is a part of the salvage pathway for re-utilizing pyridoxal 5'-phosphate, which serves as a coenzyme for dozens of enzymes involved in amino acid and sugar metabolism. Clones of two pyridoxal kinases from Escherichia coli and one from human were inserted into a pET 22b plasmid and expressed in E. coli. All three enzymes were purified to near homogeneity and kinetic constants were determined for the three vitamin substrates. Previous studies had suggested that ZnATP was the preferred trinucleotide substrate, but our studies show that under physiological conditions MgATP is the preferred substrate. One of the two E. coli kinases has very low activity for pyridoxal, pyridoxine, and pyridoxamine. We conclude that in vivo this kinase may have an alternate substrate involved in another metabolic pathway and that pyridoxal has only a poor secondary activity for this kinase.     

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.     

Transaminations catalysed by brain glutamate decarboxylase.

In addition to normal decarboxylation of glutamate to 4-aminobutyrate, glutamate decarboxylase from pig brain was shown to catalyse decarboxylation-dependent transamination of L-glutamate and direct transamination of 4-aminobutyrate with pyridoxal 5'-phosphate to yield succinic semialdehyde and pyridoxamine 5'-phosphate in a 1:1 stoichiometric ratio. Both reactions result in conversion of holoenzyme into apoenzyme. With glutamate as substrate the rates of transamination differed markedly among the three forms of the enzyme (0.008, 0.012 and 0.029% of the rate of 4-aminobutyrate production by the alpha-, beta- and gamma-forms at pH 7.2) and accounted for the differences among the forms in rates of inactivation by glutamate and 4-aminobutyrate. Rates of transamination were maximal at about pH 8 and varied in parallel with the rate constants for inactivation from pH 6.5 to 8.0. Rates of transamination of glutamate and 4-aminobutyrate were similar, suggesting that the decarboxylation step is not entirely rate-limiting in the normal mechanism. The transamination was reversible, and apoenzyme could be reconstituted to holoenzyme by reverse transamination with succinic semialdehyde and pyridoxamine 5'-phosphate. As a major route of apoenzyme formation, the transamination reaction appears to be physiologically significant and could account for the high proportion of apoenzyme in brain.     

家蚕体内维生素B6的存在形态和转换代谢

采用不含桑叶粉末、以去维生素牛乳酪蛋白为蛋白源的准合成饲料饲育家蚕Bombyx mori幼虫,探讨了家蚕体内维生素B（VB₆）化合物的存在形态和转换代谢途经。随饲料中盐酸吡哆醇（PN-HCl）添加量的增加,幼虫体内吡哆醇(PN)含量相应变化,其次是吡哆醛(PL);而辅酶型磷酸吡哆醛(PLP)和磷酸吡哆胺(PMP)含量存在稳定性。饲料中的吡哆醇以单纯扩散的形式进入体液;体液中的吡哆醇被各种组织吸收后,在各自的吡哆醛激酶和PNP/磷酸吡哆胺氧化酶的作用下,转变成辅酶型磷酸吡哆醛。家蚕不同于哺乳动物,没有特定的辅酶型磷酸吡哆醛形成组织和辅酶型磷酸吡哆醛的转送机制。同时家蚕体内缺乏具储存VB₆功能的辅酶型磷酸吡哆醛结合蛋白,推测这是用缺乏VB₆的饲料饲育各龄起蚕,幼虫当龄死亡的主要原因。     

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.     

Activation of Glutamate Apodecarboxylase by Succinic Semialdehyde and Pyridoxamine 5''-Phosphate

Glutamate apodecarboxylase was activated by incubation with succinic semialdehyde and pyridoxamine 5'-phosphate. Activation required both compounds and was highly selective for succinic semialdehyde. Of 18 analogs tested, only glyoxylate, pyruvate, oxaloacetate, and 2-oxoglutarate activated the apoenzyme significantly, but much higher concentrations of these compounds than of succinic semialdehyde were required. In the presence of pyridoxamine 5'-phosphate, the concentration of succinic semialdehyde giving half-maximal activation of apoenzyme was 7 microM. In contrast, the Ki for succinic semialdehyde as a competitive inhibitor of glutamate decarboxylation was 1.2 mM, indicating that apoenzyme with bound pyridoxamine 5'-phosphate has a much higher affinity for succinic semialdehyde than does holoenzyme. The concentration of pyridoxamine 5'-phosphate giving half-maximal activation was 17 microM, which is more than an order of magnitude greater than the corresponding value for pyridoxal 5'-phosphate.     

Identification of amino acid residues modified by pyridoxal 5'-phosphate in Escherichia coli glutamine synthetase

Chemical modification studies with pyridoxal 5'-phosphate have indicated that lysine(s) appear to be at or near the active site of Escherichia coli glutamine synthetase (Colanduoni, J., and Villafranca, J. J. (1985) J. Biol. Chem. 260, 15042-15050; Whitley, E. J., Jr., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). Enzyme samples were prepared that contained approximately 1, approximately 2, and approximately 3 pyridoxamine 5'-phosphate residues/50,000-Da monomer; the activity of each sample was 100, 25, and 14% of the activity of unmodified enzyme, respectively. Cyanogen bromide cleavage of each enzyme sample was performed, the peptides were separated by high performance liquid chromatography, and the peptides containing pyridoxamine 5'-phosphate were identified by their absorbance at 320 nm. These isolated peptides were analyzed for amino acid composition and sequenced. The N terminus of the protein (a serine residue) was modified by pyridoxal 5'-phosphate at a stoichiometry of approximately 1/50,000 Da and this modified enzyme had full catalytic activity. Beyond a stoichiometry of approximately 1, lysines 383 and 352 reacted with pyridoxal 5'-phosphate and each modification results in a partial loss of activity. When various combinations of substrates and substrate analogs (ADP/Pi or L-methionine-SR-sulfoximine phosphate/ADP) were used to protect the enzyme from modification, Lys-352 was protected from modification indicating that this residue is at the active site. Under all experimental conditions employed, Lys-47, which reacts with the ATP analog 5'-p-fluorosulfonylbenzoyl-adenosine does not react with pyridoxal 5'-phosphate.     

Vitamin B6s inhibit oxidative stress caused by Alzheimer's disease-related Cu(II)-β-amyloid complexes-cooperative action of phospho-moiety

Cu(II) complexes of Alzheimer's disease-related β-amyloid (Aβ) peptides exhibit metal-centered oxidation chemistry. The metallo-Aβ complexes are the hallmark of the disease and have been attributed to the generation of reactive oxygen species (ROS), causing oxidative stress. In this communication, the inhibitions of the oxidative activity of Cu(II)-Aβ by vitamin B6 compounds pyridoxamine (PM), pyridoxine (PN), pyridoxal (PL), and pyridoxal-5'-phosphate (PLP) are presented. These B6's are competitive inhibitors toward dopamine oxidation by Cu(II)-Aβ(1-20), with K(i) values of 1.4, 8.3, 1.2, and 0.2mM, respectively. The phospho-moiety in PLP seems to exhibit cooperative inhibition, affording a clue for future design of inhibitors.     

Pyridoxal 5'-phosphate is a selective inhibitor in vivo of DNA polymerase alpha and epsilon

Vitamin B(6) compounds such as pyridoxal 5(')-phosphate (PLP), pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM), which reportedly have anti-angiogenic and anti-cancer effects, were thought to be inhibitors of some types of eukaryotic DNA polymerases. PL moderately inhibited only the activities of calf DNA polymerase alpha (pol alpha), while PN and PM had no inhibitory effects on any of the polymerases tested. On the other hand, PLP, a phosphated form of PL, was potentially a strong inhibitor of pol alpha and epsilon from phylogenetic-wide organisms including mammals, fish, insects, plants, and protists. PLP did not suppress the activities of prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I and Taq DNA polymerase, or DNA-metabolic enzymes such as deoxyribonuclease I. For pol alpha and epsilon, PLP acted non-competitively with the DNA template-primer and competitively with the nucleotide substrate. Since PL was converted to PLP in vivo after being incorporated into human cancer cells, the anti-angiogenic and anti-cancer effects caused by PL must have been caused by the inhibition of pol alpha and epsilon activities after conversion to PLP.     

Active site structure and stereospecificity of Escherichia coli pyridoxine-5'-phosphate oxidase.

Pyridoxine-5'-phosphate oxidase catalyzes the oxidation of either the C4' alcohol group or amino group of the two substrates pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate to an aldehyde, forming pyridoxal 5'-phosphate. A hydrogen atom is removed from C4' during the oxidation and a pair of electrons is transferred to tightly bound FMN. A new crystal form of the enzyme in complex with pyridoxal 5'-phosphate shows that the N-terminal segment of the protein folds over the active site to sequester the ligand from solvent during the catalytic cycle. Using (4'R)-[(3)H]PMP as substrate, nearly 100 % of the radiolabel appears in water after oxidation to pyridoxal 5'-phosphate. Thus, the enzyme is specific for removal of the proR hydrogen atom from the prochiral C4' carbon atom of pyridoxamine 5'-phosphate. Site mutants were made of all residues at the active site that interact with the oxygen atom or amine group on C4' of the substrates. Other residues that make interactions with the phosphate moiety of the substrate were mutated. The mutants showed a decrease in affinity, but exhibited considerable catalytic activity, showing that these residues are important for binding, but play a lesser role in catalysis. The exception is Arg197, which is important for both binding and catalysis. The R197 M mutant enzyme catalyzed removal of the proS hydrogen atom from (4'R)-[(3)H]PMP, showing that the guanidinium side-chain plays an important role in determining stereospecificity. The crystal structure and the stereospecificity studies suggests that the pair of electrons on C4' of the substrate are transferred to FMN as a hydride ion.     

Purification and properties of hydroxymethylpyrimidine kinase from Escherichia coli

Hydroxymethylpyrimidine kinase, which catalyzes the conversion of 2-methyl-4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) to its monophosphate, is purified about 3300-fold to apparent homogeneity from the cell-free extracts of E. coli K-12 through four successive steps of column chromatographies. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis and its molecular weight is estimated to be 43 000-44 000. The enzyme phosphorylated each of the pyridoxine substrates, pyridoxine, pyridoxal and pyridoxamine as well as hydroxymethylpyrimidine, and the reaction gave rise to a corresponding 5'-phosphate compound. The Km values of the purified enzyme for hydroxymethylpyrimidine and for pyridoxine are 1.1.10(-4) and 6.6.10(-5) M, respectively. Pyridoxine inhibits competitively the phosphorylation of hydroxymethylpyrimidine with a Ki value of 2.7.10(-6) M and hydroxymethylpyrimidine shows the same for that of pyridoxine with a Ki value of 9.0.10(-5) M. A similarity in enzymic properties between the hydroxymethylpyrimidine kinase and an enzyme which has been characterized as pyridoxal kinase leads to the assumption that both hydroxymethylpyrimidine and pyridoxine might be phosphorylated by the same enzyme species.     

Interconversions of different forms of vitamin B6 in tobacco plants

There are six different vitamin B₆ (VB₆) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5′-phosphate (PLP), pyridoxamine 5′-phosphate (PMP), and pyridoxine 5′-phosphate (PNP), of which PLP is the active form. Although plants are a major source of VB₆ in the human diet, and VB₆ plays an important role in plants, the mechanisms underlying the interconversions of different VB₆ forms are not well understood. In this study, in vitro tobacco plants were grown on Murashige and Skoog (MS) basal media supplemented with 100 mg/L of PM, PL or PN and the abundance of the different B₆ vitamers in leaf tissue was quantified by high performance liquid chromatography (HPLC). The total amount of VB₆ was about 3.9 μg/g fresh weight of which PL, PM, PN, PLP and PMP accounted for 23%, 14%, 37%, 20% and 6%, respectively. Tobacco plants contained a trace amount of PNP. Supplementation of the culture medium with any of the non-phosphorylated vitamers resulted in an increase in total VB₆ by about 10-fold, but had very little impact on the concentrations of the endogenous phosphorylated vitamers. Administration of either PM or PN increased their endogenous levels more than the levels of any other endogenous B₆ vitamers. PL supplementation increased the levels of plant PN and PM significantly, but not that of PL, suggesting that efficient conversion pathways from PL to PN and PM are present in tobacco. Additionally, maintenance of a stable level of PLP in the plant is not well-correlated to changes in levels of non-phosphorylated forms.     

Disruption of the plr1+ gene encoding pyridoxal reductase of Schizosaccharomyces pombe

Pyridoxal (PL) reductase encoded by the plr1(+) gene practically catalyzes the irreversible reduction of PL by NADPH to form pyridoxine (PN). The enzyme has been suggested to be involved in the salvage synthesis of pyridoxal 5'-phosphate (PLP), a coenzyme form of vitamin B(6), or the excretion of PL as PN from yeast cells. In this study, a PL reductase-disrupted (plr1 Delta) strain was constructed and its phenotype was examined. The plr1 Delta cells showed almost the same growth curve as that of wild-type cells in YNB and EMM media. In EMM, the plr1 Delta strain became flocculent at the late stationary phase for an unknown reason. The plr1 Delta cells showed low but measurable PL reductase activity catalyzed by some other protein(s) than the enzyme encoded by the plr1(+) gene, which maintained the flow of "PL --> PN --> PNP --> PLP" in the salvage synthesis of PLP. The total vitamin B(6) and pyridoxamine 5'-phosphate contents in the plr1 Delta cells were significantly lower than those in the wild-type ones. The percentages of the PLP amount as to the other vitamin B(6) compounds were similar in the two cell types. The amount of PL in the culture medium of the disruptant was significantly higher than that in the wild-type. In contrast, PN was much higher in the latter than the former. The plr1 Delta cells accumulated a 6.1-fold higher amount of PL than the wild-type ones when they were incubated with PL. The results showed that PL reductase encoded by the plr1(+ )gene is involved in the excretion of PL after reducing it to PN, and may not participate in the salvage pathway for PLP synthesis.     

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.     

Bovine brain low Mr acid phosphatase: purification and properties

Low molecular weight acid phosphatase from bovine brain was purified to homogeneity using affinity chromatography on p-aminobenzylphosphonic acid-agarose to obtain the enzyme with both high specific activity (110 mumol min-1 mg-1 measured at pH 5.5 and 37 degrees C with p-nitrophenyl phosphate as substrate) and good yields. The enzyme was characterized with respect to molecular weight, amino acid composition, pH optimum, Km and Vmax in varying substrates, and to the Ki of varying inhibitors. Furthermore, transphosphorylation to glycerol was demonstrated by measuring the released p-nitrophenol/Pi concentration ratio during the initial phase of the catalyzed reaction. The enzyme was inactivated by iodoacetate and 1,2-cycloexanedione. Inorganic phosphate, a competitive inhibitor, protected the enzyme from being inactivated by the above compounds, demonstrating the involvement of both cysteine(s) and arginine(s) at the active site of the enzyme. Furthermore, the strong inhibition exerted by pyridoxal 5'-phosphate and the low inhibitory capacity possessed by the pyridoxal 5'-phosphate analogues pyridoxamine 5'-phosphate and pyridoxal, indicate that at least one lysine residue is present at the active site.     

Effect of pyridoxal 5'-phosphate on Ca2+-stimulated adenosine triphosphatase activity in microsomes of rat submandibular gland in vitro

1. The Ca2+-ATPase activity in microsomes of rat submandibular gland was inhibited by pyridoxal 5'-phosphate in vitro. 2. The dissociation constant of the enzyme-pyridoxal 5'-phosphate complex was estimated to be 6.5 mM. 3. The inhibition of pyridoxal 5'-phosphate for both ATP and Ca2+ was competitive. 4. The order of inhibitory effectiveness of pyridoxal 5'-phosphate analogs was pyridoxal 5'-phosphate greater than pyridoxal HCl greater than pyridoxamine 5'-phosphate greater than pyridoxamine HCl. 5. The enzyme-pyridoxal 5'-phosphate complex was nonreducible with sodium borohydride.