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PubMed1991

Enzymatic fluorometric assay for plasma pyridoxal 5'-phosphate.

An enzymatic fluorometric assay to quantitate plasma pyridoxal 5'-phosphate (PLP) is described. PLP is preincubated for 30 min with purified tyrosine decarboxylase apoenzyme (TDA) in acetate buffer and is then incubated with L-tyrosine for 60 min. The decarboxylated metabolite, tyramine, is extracted into ethyl acetate, air dried, dissolved in borate buffer and reacted with fluorescamine to form a fluorophor (395/475 nm). Recovery of exogenous PLP was 83%. After log-normal transformation of 129 plasma PLP levels obtained from 3 groups of subjects (in-patients, out-patients, staff), a normal distribution curve was obtained yielding a mean of 83 nmol/L. With 95% confidence level, the reference limits were 32-170 nmol/L. There were no age or sex differences in mean plasma PLP levels between any of the 3 groups of subjects studied.

Adolescent↗

PubMed1993

Similarity between serine hydroxymethyltransferase and other pyridoxal phosphate-dependent enzymes.

A structural homology of the pyridoxal-5'-phosphate (PLP)-dependent enzyme serine hydroxymethyltransferase (SHMT) with aspartate aminotransferase (AAT) is proposed. Although the two sequences are very dissimilar, a reasonable alignment was obtained using the profile analysis method. Sequences of AAT and dialkylglycine decarboxylase (DGD), for which crystal structure data are available, have been aligned on the basis of their structure superposition. A profile was then calculated and SHMT sequence aligned to it. Three of the four residues conserved in all aminotransferases (including the PLP-binding lysine) are matched. A profile search with DGD-AAT-SHMT profile is more selective and sensitive than individual sequence profiles for PLP-dependent enzyme detection. Potential homologies with the eryC1 gene product involved in erythromycin biosynthesis and with amino acid decarboxylases were observed. Homology with AAT will be used as a guideline for planning site-directed mutagenesis experiments on SHMT.

Amino Acid Sequence↗

PubMed1988

Pyridoxal 5'-phosphate levels in children with sickle cell disease.

Pyridoxal 5'-phosphate (PLP), the major coenzyme form of vitamin B6, is known to have antisickling properties in vitro. Recently, low plasma PLP levels were reported in a group of adults with sickle cell anemia. We measured the plasma PLP levels in a group of 55 asymptomatic nontransfused children with sickle cell diseases (SCD) to determine the prevalence of low plasma PLP levels in this population. Comparative studies were made with the measurement of PLP in three other groups serving as controls: Group A (black children, n = 36); Group B (white children, n = 37); and Group C (black adults, n = 13). PLP was measured directly in plasma by a radioenzymatic technique. The results of these comparisons showed that there was no statistically significant difference in plasma PLP of black children with SCD (10.7 +/- 10.0 ng/ml) as compared with black control children (group A, 9.0 +/- 12.3 ng/ml). The low plasma levels PLP in these two groups were significantly lower than that of the plasma PLP of white control children (group B, 15.85 +/- 15.92 ng/ml). This data suggest that a high prevalence of low PLP levels exists in black children seen at Grady Memorial Hospital, both with and without SCD.

Adolescent↗

PubMed1995

Pulsed electron paramagnetic resonance studies of the lysine 2,3-aminomutase substrate radical: evidence for participation of pyridoxal 5'-phosphate in a radical rearrangement.

The role of pyridoxal 5'-phosphate (PLP) in the radical-mediated amino group migration catalyzed by lysine 2,3-aminomutase from Clostridia SB4 has been investigated by electron spin echo envelope modulation (ESEEM) spectroscopy. This pulsed electron paramagnetic resonance (EPR) method was used to estimate the distance between the unpaired electron in the alpha-radical of beta-lysine, a steady-state intermediate in the reaction, and deuterium at the C4' position of the cofactor, PLP. [4'-2H]PLP was synthesized and exchanged into the enzyme. The steady-state radical was generated in the labeled samples and in samples with unlabeled PLP by addition of L-lysine.H2SO4 to activated enzyme. ESEEM spectra of the samples prepared with [4'-2H]PLP exhibited distinctive low-frequency modulations that were not present in spectra of matched samples with unlabeled PLP. Fourier transformation of the modulations yielded a prominent doublet signal centered about the Larmor frequency of deuterium. The magnitude of the doublet splitting of the 2H ESEEM signal exhibited angle selection across the CW EPR powder pattern. The observed angle selection, as well as simulation of the time domain spectra, indicated that the doublet splitting was due to the combined effects of the 2H hyperfine and nuclear quadrupole interactions. The influences of the quadrupole interaction and of isotropic and dipolar hyperfine interactions were explored by simulations of the ESEEM spectra. The analysis indicates a distance of < 3.5 A between the 2H at C4' of PLP and the radical center at C alpha lysine. The data are most compatible with an aldimine linkage between PLP and the beta-nitrogen of beta-lysine.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Isomerases↗

PubMed2000

The novel pyridoxal-5'-phosphate derivative PPNDS potently antagonizes activation of P2X(1) receptors.

Pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonat e) (PPNDS) potently antagonized P2X(1) receptor-mediated responses in rat vas deferens (pK(B)=7.43) and Xenopus laevis oocytes (pIC(50)=7. 84). It showed lower (up to 20,000-fold) inhibitory potency on ecto-nucleotidase in Xenopus oocytes and on P2Y(1) receptors in guinea-pig ileum (pA(2)=6.13). PPNDS did not interact with alpha(1A)-adrenoceptors, adenosine A(1) and A(2B), histamine H(1) and muscarinic M(3) receptors. Thus, PPNDS is a novel, specific P2 receptor antagonist and represents the pyridoxal-5'-phosphate derivative with the highest potency at P2X(1) receptors.

Animals↗

PubMed2003

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.

Cell Survival↗

PubMed1984

Substrate-cofactor interactions for glycogen phosphorylase b: a binding study in the crystal with heptenitol and heptulose 2-phosphate.

The structural relationships between substrate and pyridoxal phosphate in glycogen phosphorylase b (EC 2.4.1.1) have been studied by X-ray diffraction experiments at 3-A resolution. Recent work [Klein, H. W., Im, M. J., & Helmreich, E. J. M. (1984) in Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos, A. E., Ed.) pp 147-160, Liss, New York] has shown that phosphorylase in the presence of inorganic phosphate catalyzes the conversion of heptenitol to heptulose 2-phosphate. The latter compound is a dead-end product and a most potent inhibitor (Ki = 14 microM). The X-ray diffraction studies show that heptenitol binds at the catalytic site of phosphorylase in a position essentially identical with that observed for the glucopyranose moiety of glucose 1-phosphate. Incubation of a phosphorylase b crystal for 50 h in a solution containing the substrates heptenitol and inorganic phosphate and the activators AMP and maltohetaose resulted in the formation of a phosphorylated product bound at the active site. The structure of this product, as analyzed by a difference Fourier synthesis at 3 A, is consistent with that of heptulose 2-phosphate. Analysis of the surrounding soak solution by thin-layer chromatography showed that heptulose 2-phosphate was produced under these conditions. Heptulose 2-phosphate binds with its glucopyranose moiety in the same position as that for glucose 1-phosphate, but there is a marked difference in phosphate positions. The presence of the methyl group in the beta-configuration in heptulose 2-phosphate forces a change in the torsion angle O5-C1-O1-P from 117 degrees as observe in glucose 1-phosphate to -136 degrees in heptulose 2-phosphate. The "down" position of the phosphate (with respect to the crystallographic z axis) results in a change in the distance between the 5'-phosphorus atom of the pyridoxal phosphate and the phosphorus atom of the substrate from 6.8 (with glucose 1-phosphate) to 4.5 A (with heptulose 2-phosphate). The closest distance between the phosphate oxygen of the cofactor and a phosphate oxygen of heptulose 2-phosphate is 2.7 A, and it is assumed that there must be a hydrogen bond between them. These observations are consistent with the NMR experiments reported in the preceding paper in which sharing of a proton between heptulose 2-phosphate and pyridoxal 5'-phosphate is observed [Klein, H.W., Im, M. J., Palm, D., & Helmreich, E. J. M. (1984) Biochemistry (preceding paper in this issue)].(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Monophosphate↗

PubMed1976

Structural and functional interdependence of the protomers of Escherichia coli K 12 tryptophanase during binding of pyridoxal 5'-phosphate.

It has been shown previously that the binding of pyridoxal 5'-phosphate to Escherichia coli K 12 tryptophanase brings about an important conformational change of the protein. The way in which this structural change is transmitted from holoprotomers to apoprotomers is investigated here, using hybrid molecules (between apoprotomers and irreversibly saturated holoprotomers). It is shown that the binding of two or three coenzyme molecules per tetramer stabilizes the whole molecule against thermal inactivation and cold-induced dissociation. The change in conformation induced on an apoprotomer by the proximity of three holoprotomers is described, using three structural probes: the kinetics of binding of the cofactor and of 5'-phosphopyridoxyl-tryptophan (an analog of an intermediate in the catalytic reaction), and the reactivity of the essential cysteines. The kinetic anticooperatively in the binding of pyridoxal 5'-phosphate is confirmed, some of its parameters are determined, and its mechanism is interpreted in relation to the coupling between the protomers.

Apoenzymes↗

PubMed1994

Pyridoxal 5'-phosphate related changes in retention of 1,25-dihydroxy vitamin D-receptor ligands in rat intestinal mucosa cell nuclei.

After feeding rats a vitamin B-6-deficient diet, we observed a decrease in pyridoxal 5'-phosphate concentrations in intestinal mucosa cells to 32 and 48% of control in cytoplasm and cell nuclei, respectively. Correlation analysis suggested that there were two pyridoxal 5'-phosphate pools in the nuclei: a "mobile" pool (equivalent to about 5% the concentration of the cytoplasmic pyridoxal 5'-phosphate), and a "stable" pool, which was independent of cytoplasmic fluctuations of pyridoxal 5'-phosphate (about 9 pmol pyridoxal 5'-phosphate/mg DNA). Reduction in pyridoxal 5'-phosphate content in the cells of vitamin B-6-deficient animals was accompanied by a substantial increase in 1,25-dihydroxyvitamin D-receptor ligand concentration in the cell nuclei (76.6 +/- 19.7 vs 762 +/- 291 fmol/mg DNA, mean +/- SEM). The degree of 1,25-dihydroxyvitamin D accumulation in the nuclei appeared to be an exponential function of the "mobile" nuclear pyridoxal 5'-phosphate concentration. Semilogarithmic transformation of the data yielded a straight line, representing an inverse correlation between the cytoplasm-related nuclear pool of pyridoxal 5'-phosphate and the logarithm of the 1,25-dihydroxyvitamin D concentration in the nuclei (r = -0.95). These data suggest that pyridoxal 5'-phosphate may be related to 1,25-dihydroxyvitamin D retention in the nuclei, possibly through interaction of the pyridoxal 5'-phosphate with the vitamin D receptor protein in the nuclei.

Alkaline Phosphatase↗

PubMed2001

Seizures induced by intracerebral administration of pyridoxal-5'-phosphate: effect of GABAergic drugs and glutamate receptor antagonists.

Pyridoxal-5'-phosphate (PLP), the cofactor of glutamate decarboxylase, paradoxically induces convulsions when injected intracranially in adult mammals. We have tested the effect of some GABAergic and antiglutamatergic drugs on the behavioral and electroencephalographic (EEG) seizures produced by intracerebroventricular (i.c.v.) microinjection of 1 micromol PLP in the rat. PLP induced barrel turning, running fits and tonic-clonic convulsions, which started 5-10 min after recovery from the anesthesia (halothane), peaked at 20 min and disappeared at about 50 min. These symptoms were accompanied by frequent high amplitude EEG spike burst discharges. Pyridoxal, pyridoxamine-5'-phosphate or deoxypyridoxine were ineffective. The i.c.v. microinjection of the GABAergic compounds muscimol, isoguvacine, aminooxyacetic acid or GABA itself, significantly protected against PLP effects. In contrast, the NMDA receptor antagonists MK-801 and the non-NMDA receptor antagonist NBQX, failed to protect and induced motor alterations and mortality. We conclude that a temporary decrease of the GABA(A) receptor function is involved in the convulsant effect of PLP. This decrease might be due to the formation of a Schiff base between the carbonyl group of PLP and the epsilon-amino group of a functionally crucial lysine residue located in one extracellular loop of the GABA(A) receptor.

Animals↗

PubMed2003

Plasma pyridoxal 5'-phosphate concentration is correlated with functional vitamin B-6 indices in patients with rheumatoid arthritis and marginal vitamin B-6 status.

Many patients with rheumatoid arthritis (RA) have low plasma pyridoxal-phosphate (PLP) but a normal erythrocyte aspartate aminotransferase activity coefficient (alpha EAST), a measure of vitamin B-6 status in the erythrocytes, compared with healthy subjects. The goal of the present study was to examine the correlations of PLP levels in these two compartments (plasma and erythrocytes) with other established indices of vitamin B-6 status, and to determine which indicator better reflects functional status of vitamin B-6 in patients with RA. Multiple indices of vitamin B-6 status were measured in 33 patients with RA. Plasma PLP, urinary 4-pyridoxic acid (4-PA), net increase in plasma total homocysteine after a methionine load (DeltatHcy) and net increase in urinary xanthurenic acid after a tryptophan load (DeltaXA) were log-transformed to reach normality for statistical analyses. We found that log-plasma PLP levels were inversely correlated with both log-DeltatHcy (r = -0.368, P = 0.035) and log-DeltaXA (r = -0.333, P = 0.05). Plasma PLP was not correlated with alpha EAST or urinary 4-PA excretion. In contrast, erythrocyte PLP was inversely correlated with alpha EAST (r = -0.431, P = 0.012) and positively correlated with log-4-PA (r = 0.475, P = 0.005), but erythrocyte PLP was not correlated with the outcomes of a methionine or tryptophan load test. Erythrocyte PLP and log-4-PA, but not plasma PLP, were correlated with dietary intake of vitamin B-6 after adjusting for protein intake (r = 0.420, P = 0.015 and r = 0.333, P = 0.05, respectively). We suggest that in patients with RA, plasma PLP levels are a better diagnostic indicator of functional vitamin B-6 status than erythrocyte PLP levels.

Adult↗

PubMed1997

Crystallographic investigation of the dependence of calcium and phosphate ions for notexin.

The crystal structure of the neurotoxic phospholipase A2, notexin, revealed three binding sites for sulphate ions which were suggested to be phosphate binding sites of importance for the activity of the toxin. The present investigation shows that the sulphate ion bound to the major binding site alters the structure of residues 60-75. In the absence of sulphate and phosphate, the structure of this loop has a conformation which partly resembles the non-neurotoxic PLA2s. The affinity of notexin for phosphate is 17 microM, as measured by the increase in fluorescence at 345 nm. Since the concentrations of phosphate and sulphate ions in blood plasma are 3 and 1 mM, respectively, the binding site must be occupied under physiological conditions. This major sulphate/phosphate binding site explains the specific affinity labelling by pyridoxal phosphate. Pyridoxal phosphate binds to this anion binding site which allows the reaction with Lys-88 or Lys-89. The structure of notexin in the presence and absence of Ca2+ shows only small local structural differences.

Binding Sites↗

PubMed1989

A simple liquid-chromatographic method for measuring vitamin B6 compounds in plasma.

This relatively simple high-performance liquid chromatographic (HPLC) method for measuring all seven known forms of vitamin B6 in plasma from individuals supplemented with pyridoxine hydrochloride shows good analytical recovery (85-98%) and precision. Within-run and between-run CVs for plasmas supplemented with standards were 4% and 7%, respectively. The major forms of B6 found in unsupplemented plasma from normal subjects were pyridoxal phosphate and 4-pyridoxic acid, with pyridoxal just detectable. The HPLC procedure correlated well (r = 0.94) with a modification of an enzymatic method involving apotryptophanase (Anal Biochem 1972;45:567-76) for measuring plasma pyridoxal phosphate, and also (r = 0.94) with a routine method for determining 4-pyridoxic acid in urine (Clin Chem 1964;10:479-89). Elimination of pyridoxine from the plasma of both normal and hyperoxaluric individuals was shown to be very rapid, with half-lives (t1/2) of 45 and 40 min, respectively. Finally, we present evidence for the existence of two other forms of B6 and discuss the possibility of a new metabolic pathway in vitamin B6 metabolism.

Chromatography, High Pressure Liquid↗

PubMed1990

Chemical modification by pyridoxal 5'-phosphate and cyclohexane-1,2-dione indicates that Lys-7 and Arg-10 are involved in the p2 phosphate-binding subsite of bovine pancreatic ribonuclease A.

Steric and chemical evidence had previously shown that residues Lys-7 and/or Arg-10 of bovine pancreatic RNAase A could belong to the p2 phosphate-binding subsite, adjacent to the 3' side of the main site p1. In the present work chemical modification of the enzyme with pyridoxal 5'-phosphate and cyclohexane-1,2-dione was carried out in order to identify these residues positively as part of the p2 site. The reaction with pyridoxal 5'-phosphate yields three monosubstituted derivatives, at Lys-1, Lys-7 and Lys-41. A strong decrease in the yield of derivatives at Lys-7 and Lys-41 was observed when either p1 or p2 was specifically blocked by 5'-AMP or 3'-AMP respectively. These experiments indicate that both sites are needed for the reaction of pyridoxal 5'-phosphate with RNAase A to take place. The positive charge in one of the sites interacts with the phosphate group of pyridoxal 5'-phosphate, giving the proper orientation to the carbonyl group, which then reacts with the lysine residue present in the other site. The absence of reaction between pyridoxal 5'-phosphate and an RNAase derivative that has the p2 site blocked supports this hypothesis. Labelling of Lys-7 with pyridoxal 5'-phosphate has a more pronounced effect on the kinetics with RNA than with the smaller substrate 2',3'-cyclic CMP. In addition, when the phosphate moiety of the 5'-phosphopyridoxyl group was removed with alkaline phosphatase the kinetic constants with 2',3'-cyclic CMP returned to values very similar to those of the native enzyme, whereas a higher Km and lower Vmax. were still observed for RNA. This indicates that this new derivative has recovered a free p1 site and, hence, the capability to act on 2',3'-cyclic CMP, but the presence of the pyridoxyl group bound to Lys-7 is still blocking a secondary phosphate-binding site, namely p2. Finally, reaction of cyclohexane-1,2-dione at Arg-10 is suppressed in the presence of 3'-AMP but only a 19% decrease is observed with 5'-AMP, suggesting that Arg-10 is also close to the p2 phosphate-binding subsite.

Animals↗

PubMed2004

Pyridoxal 5'-phosphate inactivates DNA topoisomerase IB by modifying the lysine general acid.

The present results demonstrate that pyridoxal, pyridoxal 5'-phosphate (PLP) and pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) inhibit Candida guilliermondii and human DNA topoisomerases I in forming an aldimine with the epsilon-amino group of an active site lysine. PLP acts as a competitive inhibitor of C.guilliermondii topoisomerase I (K(i) = 40 microM) that blocks the cleavable complex formation. Chemical reduction of PLP-treated enzyme reveals incorporation of 1 mol of PLP per mol of protein. The limited trypsic proteolysis releases a 17 residue peptide bearing a lysine-bound PLP (KPPNTVIFDFLGK*DSIR). Targeted lysine (K*) in C.guilliermondii topoisomerase I corresponds to that found in topoisomerase I of Homo sapiens (K532), Candida albicans (K468), Saccharomyces cerevisiae (K458) and Schizosaccharomyces pombe (K505). In the human enzyme, K532, belonging to the active site acts as a general acid catalyst and is therefore essential for activity. The spatial orientation of K532-PLP within the active site was approached by molecular modeling using available crystallographic data. The PLP moiety was found at close proximity of several active residues. PLP could be involved in the cellular control of topoisomerases IB. It constitutes an efficient tool to explore topoisomerase IB dynamics during catalysis and is also a lead for new drugs that trap the lysine general acid.

Adenosine Diphosphate↗

PubMed1978

Levodopa-mediated alteration in the activity of pyridoxal kinase in rat basal ganglia.

The acute intraperitoneal administration of 400 mg/kg of levodopa reduced the concentration of pyridoxal phosphate and increased the activity of pyridoxal kinase. The chronic oral administration of 100 mg/kg/day of levodopa did not alter the concentration of pyridoxal phosphate, but increased the activity of pyridoxal kinase in 7 days. This initial elevation in kinase activity returned to normal levels in 2 weeks, became reduced in 3 weeks and then fluctuated back to normal levels in 4 weeks. These results are interpreted to indicate that chronic intake of levodopa results in adaptive alteration in the activity of pyridoxal kinase. This maintains the steady-state concentration of pyridoxal phosphate in order to meet the levodopa-induced enhancement in decarboxylation reaction.

Animals↗

PubMed1979

Determination of serum aminotransferases: activation by pyridoxal-5'-phosphate in relation to substrate concentration.

To investigate the activation of aspartate- and alanine aminotransferases by pyridoxal-5'-phosphate, we determined the enzymatic activity in serum in two different ways: (a) Preincubation of the serum alone or the serum with pyridoxal-5'-phosphate and starting the reaction by the addition of the serum sample or the serum sample + coenzyme, respectively. (b) Preincubation of the serum or the serum with pyridoxal-5'-phosphate in the reaction medium and starting the reaction by adding 2-oxoglutarate. There are only small differences in activities of both aminotransferases determined according to these two different methods. The stimulation by pyridoxal-5'-phosphate is also of the same order, when both methods are compared. Further, these enzymatic activities were measured with use of various concentrations of substrates. From our experiments we conclude that the degree of stimulation of the apoenzyme of the two enzymes is independent of which way the enzymatic reaction is carried out or the substrate concentration, except that aspartate aminotransferase activity is more stimulated by the coenzyme at higher 2-oxoglutarate concentrations.

Alanine Transaminase↗

PubMed1983

L-serine binds to arginine-148 of the beta 2 subunit of Escherichia coli tryptophan synthase.

Inactivation of the beta 2 subunit and of the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli by the arginine-specific dicarbonyl reagent phenylglyoxal results from modification of one arginyl residue per beta monomer. The substrate L-serine protects the holo beta 2 subunit and the holo alpha 2 beta 2 complex from both inactivation and arginine modification but has no effect on the inactivation or modification of the apo forms of the enzyme. This result and the finding that phenylglyoxal competes with L-serine in reactions catalyzed by both the holo beta 2 subunit and the holo alpha 2 beta 2 complex indicate that L-serine and phenylglyoxal both bind to the same essential arginyl residue in the holo beta 2 subunit. The apo beta 2 subunit is protected from phenylglyoxal inactivation much more effectively by phosphopyridoxyl-L-serine than by either pyridoxal phosphate or pyridoxine phosphate, both of which lack the L-serine moiety. The phenylglyoxal-modified apo beta 2 subunit binds pyridoxal phosphate and the alpha subunit but cannot bind L-serine or L-tryptophan. We conclude that the alpha-carboxyl group of L-serine and not the phosphate of pyridoxal phosphate binds to the essential arginyl residue in the beta 2 subunit. The specific arginyl residue in the beta 2 subunit which is protected by L-serine from modification by phenyl[2-14C]glyoxal has been identified as arginine-148 by isolating a labeled cyanogen bromide fragment (residues 135-149) and by digesting this fragment with pepsin to yield the labeled dipeptide arginine-methionine (residues 148-149). The primary sequence near arginine-148 contains three other basic residues (lysine-137, arginine-141, and arginine-150) which may facilitate anion binding and increase the reactivity of arginine-148. The conservation of the arginine residues 141, 148, and 150 in the sequences of tryptophan synthase from E. coli, Salmonella typhimurium, and yeast supports a functional role for these three residues in anion binding. The location and role of the active-site arginyl residues in the beta 2 subunit and in two other enzymes which contain pyridoxal phosphate, aspartate aminotransferase and glycogen phosphorylase, are compared.

Amino Acid Sequence↗