Enzyme-bound Pyridoxal Phosphate Research Articles

Regulation of serine racemase activity by amino acids

The effects of various amino acids on the activity of serine racemase, purified from mouse brain, were examined. Those acting as inhibitors included compounds with electron withdrawing groups on the β-carbon of alanine (β-halo-alanines and l-serine- O-sulfate), which can act as enzyme-activated inhibitors, and compounds containing β-SH groups (cysteine and homocysteine) which react with enzyme-bound pyridoxal phosphate to form thiazolidine derivatives. Glycine and a series of metabolites related to l-aspartic acid ( l-aspartic acid, l-asparagine, and oxaloacetic acid) were also found to be competitive inhibitors of the racemase. The K i values for glycine and aspartic acid inhibition were 0.15 and 1.9 mM, respectively, indicating that alterations in the concentrations of these amino acids might play a role in the regulation of d-serine synthesis.

3-Amino-5-hydroxybenzoic Acid Synthase, the Terminal Enzyme in the Formation of the Precursor of mC7N Units in Rifamycin and Related Antibiotics

The biosynthesis of ansamycin antibiotics, like rifamycin B, involves formation of 3-amino-5-hydroxybenzoic acid (AHBA) by a novel variant of the shikimate pathway. AHBA then serves as the starter unit for the assembly of a polyketide which eventually links back to the amino group of AHBA to form the macrolactam ring. The terminal enzyme of AHBA formation, which catalyzes the aromatization of 5-deoxy-5-amino-3-dehydroshikimic acid, has been purified to homogeneity from Amycolatopsis mediterranei, the encoding gene has been cloned, sequenced, and overexpressed in Escherichia coli. The recombinant enzyme, a (His)6 fusion protein, as well as the native one, are dimers containing one molecule of pyridoxal phosphate per subunit. Mechanistic studies showed that the enzyme-bound pyridoxal phosphate forms a Schiff's base with the amino group of 5-deoxy-5-amino-3-dehydroshikimic acid and catalyzes both an alpha, beta-dehydration and a stereospecific 1,4-enolization of the substrate. Inactivation of the gene encoding AHBA synthase in the A. mediterranei genome results in loss of rifamycin formation; production of the antibiotic is restored when the mutant is supplemented with AHBA.

The Function of Arginine 363 as the Substrate carboxyl‐binding Site in Escherichia coli Serine Hydroxymethyltransferase

Both the highly conserved Arg363 and Arg372 residues of Escherichia coli serine hydroxymethyltransferase were changed to alanine and lysine residues. Each of the four mutant proteins were purified to homogeneity and characterized with respect to spectral properties of the enzyme-bound pyridoxal phosphate and kinetic properties with substrates and substrate analogs. The R372A and R372 K mutant enzymes exhibited spectra and kinetic properties close to those of the wild-type enzyme. The R363 K mutant enzyme exhibited only 0.03% of the catalytic activity of the wild-type enzyme and a 15-fold reduction in affinity for glycine and serine. The R363A mutant enzyme did not bind serine and glycine and showed no activity with serine as the substrate. Both R363 K and R363A enzymes bound amino acid esters at the active site and catalyzed the retro-aldol cleavage of serine ethyl ester and serinamide. The catalytic activity of the R363 K and R363A enzymes with the serine ethyl ester were about 0.006% and 0.1% of wild-type enzyme activity with serine, respectively. The R363A mutant enzyme catalyzed the half transamination of D-alanine methyl ester and L-alanine methyl ester at rates similar to the rates of transamination of D-alanine and L-alanine by the wild-type enzyme. The results are interpreted to show that R363 is the binding site of the amino acid substrate carboxyl group and that forming an ion pair between R363 and the substrate carboxyl group is an important feature in catalysis by serine hydroxymethyltransferase. Evidence is also provided that R363 may play a role in the substrate-induced open to closed conformational change of the active site.

Escherichia coli maltodextrin phosphorylase: contribution of active site residues glutamate-637 and tyrosine-538 to the phosphorolytic cleavage of .alpha.-glucans

The role of Escherichia coli maltodextrin phosphorylase (EC 2.4.1.1) active site residues Glu637 and Tyr538 which line the sugar-phosphate contact region of the enzyme was investigated by site-directed mutagenesis. Substitution of Glu637 by an Asp or Gln residue reduced kcat to approximately 0.2% of wild-type activity, while the Km values were affected to a minor extent. This indicated participation of Glu637 in transition-state binding rather than in ground-state binding. 31P NMR analysis of the ionization state of enzyme-bound pyridoxal phosphate suggested that Glu637 is also involved in modulation of the protonation state of the coenzyme phosphate observed during catalysis. Despite loss of proposed hydrogen-bonded substrate contacts, the Tyr538Phe mutant enzyme retained more than 10% activity; the apparent affinity of all substrates was slightly decreased. Mutations at either site affected the error rate of the enzyme (ratio of hydrolysis/phosphorolysis). Besides a role in substrate binding, the hydrogen-bond network of Tyr538 supports the exclusion of water from the active site.

Affinity purification and properties of rat liver mitochondrial l-alanine:4,5-dioxovalerate transaminase and its inhibition by hemin

The present study describes a new rapid procedure for purification of l-alanine:4,5-dioxovalerate transaminase from rat liver mitochondria which was purified 243-fold with a 32% yield to apparent homogeneity. The purification procedure involved protamine sulfate treatment, followed by phenyl-Sepharose CL-4B column chromatography and alanine-Sepharose 4B affinity chromatography. The K m values for l-alanine and 4,5-dioxovalerate were 3.3 and 0.28 m m, respectively. The enzyme-bound pyridoxal phosphate content was estimated to be two molecules per enzyme molecule. The purified enzyme was inhibited by the reaction product pyruvic acid, substrate analog, methylglyoxal, and sulfhydryl inhibitors. Excess concentrations of 4,5-dioxovalerate was also found to inhibit the enzyme and our experiments failed to demonstrate reversibility of the reaction. Only hemin among the intermediate compounds of heme metabolism tested was shown to be an inhibitor of purified alanine:4,5-dioxovalerate transaminase. Hemin was further shown as an uncompetitive inhibitor of both alanine and dioxovalerate.

Properties of a serine hydroxymethyltransferase in which an active site histidine has been changed to an asparagine by site-directed mutagenesis.

Histidine 228 at the active site of Escherichia coli serine hydroxymethyltransferase was replaced with an asparagine. The mutant enzyme was expressed in a strain of E. coli that lacks wild type enzyme. Absorption spectra, circular dichroism spectra, and differential scanning calorimetry thermograms suggest that the amino acid change at the active site causes no detectable change in the tertiary structure of the enzyme. Kinetic studies demonstrated that kcat for the mutant enzyme is about 25% of the value for the wild type enzyme with either L-serine or allothreonine as substrate. Km or Kd values for amino acid substrates and reduced folate compounds were 2-10-fold larger with the mutant enzyme. The rate of interconversion of several enzyme-glycine complexes showed that the conversion of the external aldimine to the quinoid complex is not the rate-determining step for either the mutant or wild type enzyme in the presence of tetrahydrofolate. The binding of L-serine to the wild type enzyme gives a more thermally stable enzyme and increases its affinity for tetrahydrofolate. These effects are not found when L-serine binds to the mutant enzyme. The studies demonstrate that histidine 228 is not a catalytically essential residue and suggest that it is involved in interacting with either the amino acid substrate or the enzyme-bound pyridoxal phosphate.

Photoinactivation and photoaffinity labeling of tryptophan synthase alpha 2 beta 2 complex by the product analogue 6-azido-L-tryptophan.

The photoaffinity reagent 6-azido-L-tryptophan was synthesized by chemical methods. It binds reversibly in the dark to the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli and forms a quinonoid intermediate with enzyme-bound pyridoxal phosphate (lambda max = 476 nm). The absorbance of this chromophore has been used for spectrophotometric titrations to determine the binding of 6-azido-L-tryptophan (the half-saturation value [S]0.5 = 6.3 microM). Photolysis of the quinonoid form of the alpha 2 beta 2 complex results in time-dependent inactivation of the beta 2 subunit but not of the alpha subunit. The extent of photoinactivation is directly proportional to the absorbance at 476 nm of the quinonoid intermediate prior to photolysis. The substrate L-serine is a competitive inhibitor of 6-azido-L-tryptophan binding and photoinactivation. The competitive inhibitors L-tryptophan, D-tryptophan, and oxindolyl-L-alanine also protect against photoinactivation. The results demonstrate that 6-azido-L-tryptophan is a quasi-substrate for the alpha 2 beta 2 complex of tryptophan synthase and that photolysis of the enzyme-quasi-substrate quinonoid intermediate results in photoinactivation. The modified alpha 2 beta 2 complex retains its ability to bind pyridoxal phosphate and to cleave indole-3-glycerol phosphate, a reaction catalyzed by the alpha subunit. 6-Azido-L-tryptophan (side-chain 1,2,3-14C3 labeled) was synthesized enzymatically from 6-azidoindole and uniformly labeled L-[14C]serine by the alpha 2 beta 2 complex of tryptophan synthase on a preparative scale and has been isolated. Incorporation of 14C label from 6-azido-L-[14C]tryptophan is stoichiometric with inactivation. Our finding that most of the incorporated 14C label is bound in an unstable linkage suggests that an active site carboxyl residue is the major site of photoaffinity labeling by 6-azido-L-tryptophan.

The role of glyoxylate in the regulation of biodegradative threonine dehydratase of Escherichia coli.

The activity of biodegradative threonine dehydratase of Escherichia coli K12 was reversibly inhibited by glyoxylate in the presence of AMP. Kinetic analysis showed that the inhibition was mixed with respect to L-threonine and competitive in terms of AMP; the inhibitory effect of glyoxylate was less pronounced at high protein concentrations. Incubation of dehydratase with L-threonine shifted the absorption maximum of the enzyme-bound pyridoxal phosphate from 413 to 425 nm; addition of glyoxylate completely prevented the threonine-mediated spectral shift. In addition to the inhibitory effect, incubation of purified enzyme with glyoxylate resulted in a progressive, irreversible inactivation of the enzyme and formation of inactive protein aggregates. The rates of inactivation were decreased with increasing concentrations of protein and AMP. During inactivation by glyoxylate, the 413-nm absorption maximum of the native enzyme was replaced by a new peak at 385 nm. Experiments with [14C]glyoxylate showed a rapid binding of 1 mol of glyoxylate per 147,000 g followed by a slow binding of 3 additional mol of glyoxylate; the glyoxylate-protein linkage was stable to acid precipitation and protein denaturants. Competition binding experiments revealed that pyruvate (which also inactivated the E. coli enzyme, Feldman, D.A., and Datta, P. (1975) Biochemistry 14, 1760-1767) did not interfere with the binding of glyoxylate or vice versa, suggesting that the two keto acids may occupy separate sites on the enzyme molecule. Nevertheless, experiments on enzyme inactivation using glyoxylate plus pyruvate reveal mutual interactions between these ligands in terms of lack of additive effect, retardation in the spectral shift due to glyoxylate, and stabilization of the enzyme in the presence and absence of AMP. We conclude from these results that the control of biodegradative threonine dehydratase is governed by a complex set of regulatory events resulting from reversible and irreversible association of these effectors with the enzyme molecule.

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-oxglutarate 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

Purification and characterization of methioninase from Pseudomonas putida.

Methioninase of Pseudomonas putida was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, with a specific activity 270-fold higher than that of the crude extract. 1. The purified enzyme had an S20,w of 8.37, a molecular weight of 160,000, and an isoelectric point of 5.6. 2. A break in the Arrhenius plot was observed at 40 degrees and the activation energies below and above this temperature were 15.5 and 2.97 kcal per mole, respectively. 3. In addition to L-methionine, various S-substituted derivatives of homocysteine and cysteine could serve as substrates. D-Methionine, 2-oxo-4-methylthiobutanoate, and related non sulfur-containing amino acids were inert. Equimolar formation of alpha-ketobutyrate and CH3SH was observed with methionine as a substrate. 4. In addition to the protein peak at 278 nm, two absorption maxima were observed at 345 and 430 nm at pH 7.5. Hydroxylamine removed the enzyme-bound pyridoxal phosphate, resulting in almost complete resolution with the concomitant disappearance of both peaks. Reconstruction of the treated enzyme could be achieved by addition of the cofactor; the Km value was calculated to be 0.37 muM. 5. The reported purified enzyme should be designated as L-methionine methanethiollyase (deaminating).

The mechanism of inhibition of pig-plasma benzylamine oxidase by the copper-chelating reagent cuprizone.

Treatment of pig‐plasma benzylamine oxidase with the copper‐chelating reagent cuprizone leads to the reversible formation of an enzyme · cuprizone complex with a dissociation constant of about 3 mM, followed by an irreversible conversion of this complex into an enzyme species exhibiting a new absorption band centered around 350 nm. The latter enzyme species shows no reactivity towards substrate or carbonyl reagents such as phenylhydrazine. One mole cuprizone per mole protein is sufficient to complete the chromophoric changes and to inactive the enzyme completely. Cuprizone reacts with free pyridoxal phosphate under formation of a reaction product showing maximum absorption at 350 nm in the visible region, and competes with phenylhydrazine for the protein‐bound pyridoxal phosphate in benzylamine oxidase. Cuprizone does not extract any significant amounts of copper from benzylamine oxidase, but affects electron paramagnetic resonance spectra of the enzyme and its phenylhydrazone derivative in a way suggesting a direct interaction with at least one of the two copper ions present in the protein. The latter interaction does not appear to be identical with those involved in the formation of the inactive enzyme species absorbing at 350 nm. It is concluded that the inhibitory action of cuprizone cannot be attributed to its copper‐chelating properties, but reflects an interaction with enzyme‐bound pyridoxal phosphate through a mechanism analogous to that governing Schiff‐base or hydrazone formation between enzyme and substrates or hydrazine derivatives.

Kinetics of the Interaction between Pig-Plasma Benzylamine Oxidase and Hydrazine Derivatives

1 The interaction between hydrazines and pyridoxal phosphate in pig plasma benzylamine oxidase has been studied by stopped-flow techniques. The rapid formation of chromophoric enzyme-inhibitor complexes follows second-order kinetics and rate constants for a number of hydrazine derivatives are reported. Reaction rates with enzyme-bound pyridoxal phosphate are 20–1000 times higher than with free pyridoxal phosphate. 2 Analysis of the pH-dependence of second-order rate constants for the formation of enzyme-inhibitor complexes shows that the protonated form of the hydrazines is non-reactive, and that the reaction is dependent upon protonation of an ionizing group in the enzyme with a pKa close to 9. 3 The second-order reaction between enzyme and phenylhydrazine is competitive with the second-order reduction of the enzyme by substrate, supporting the idea that Schiff-base formation between substrate and enzyme-bound pyridoxal phosphate is an obligatory step in the catalytic mechanism. Hence it follows that the formation of enzyme-inhibitor complexes may be considered as a model for the interaction between substrates and the prosthetic group of the enzyme. 4 The interaction between enzyme and phenylhydrazine does not result in any pronounced electron paramagnetic spectral changes, indicating that the spectral changes observed on reduction of the enzyme by substrate cannot be ascribed to the formation of an enzyme-substrate complex.

Regulation of Biodegradative Threonine Deaminase: III. EFFECTS OF β SUBSTITUENTS OF SUBSTRATE ON ABSORPTION SPECTRUM AND CIRCULAR DICHROISM OF THE ENZYME-BOUND PYRIDOXAL PHOSPHATE

Abstract In order to elucidate whether or not the spectral and circular dichroic changes observed during the biodegradative threonine deaminase reaction are caused by the Schiff base formation between the enzyme-bound pyridoxal phosphate and the dehydrated intermediate, the role of substituents and the absolute configuration of the β carbon of substrates was investigated using various analogs of l-threonine. The enzyme was found to catalyze the α,β elimination of l-allothreonine, dl-threo- and dl-erythro-β-hydroxynorvaline, dl-threo- and dl-erythro-β-phenylserine, dl-β-hydroxy-valine, l-β-chlorobutyrine, and l-β-chloroalanine in addition to l-threonine and l-serine. When l-threonine or l-β-chlorobutyrine was used as substrate, the absorption maximum of the enzyme-bound pyridoxal phosphate shifted from 415 nm to 434 and 442 nm, respectively, but it remained unchanged when l-allothreonine was used as substrate. l-β-Chloroalanine also caused a bathochromic shift of the absorption maximum, but l-serine did not induce such a shift. The positive circular dichroism of the enzyme-bound pyridoxal phosphate at 415 nm disappeared upon the addition of l-threonine or l-β-chloroalanine. On the other hand, l-β-chlorobutyrine did induce a negative circular dichroism during the reaction, and l-allothreonine slightly diminished the magnitude of the positive circular dichroism. These results indicate that the substituents as well as the absolute configuration of the β carbon of the substrate participate in the spectral and circular dichroic changes observed during the reaction. Thus, the shift of the absorption maximum and the circular dichroic change at 415 nm appear to occur in the step prior to the β elimination reaction. This conclusion was confirmed by a kinetic analysis of the spectral changes.

Association-Dissociation Properties of Sodium Borohydride-reduced Phosphorylase b

Sodium borohydride-reduced phosphorylase b dissociates completely to a monomeric form in 6.7% formamide, as determined by sedimentation velocity and equilibrium methods. No dissociation of the native enzyme occurs under these same conditions. A slow inactivation of NaBH4-reduced phosphorylase b occurs in formamide, and the degree of inactivation in different formamide concentrations seems related to the extent of dissociation. Removal of formamide by dialysis restores the enzymic activity and subunit structure. Results of several experiments suggest that the monomeric form is catalytically active. Approximately 30% activity remains after 1 hour of incubation in 6.7% formamide. After this stage, little change of enzymic activity occurs even after 7 hours of incubation. After 1 hour, sedimentation velocity experiments show that a single component is present with an s20,w of 5.6 S, indicating that dissociation is complete at this time. No change in s20,w was observed in the presence of substrates, glucose 1-phosphate, amylodextrin, and activator, AMP, indicating that reassociation of the monomer does not occur under conditions that closely approximate those used in the assay. Kinetic studies showed that the affinities of the enzyme with respect to its substrates and activator were reduced markedly by formamide. No homotropic interactions were seen with respect to AMP for the reduced enzyme incubated in formamide, further indicating the presence of an active monomer. Formamide affects the spectral properties of enzyme-bound pyridoxal phosphate in NaBH4-reduced phosphorylase b.

Mechanism of inhibition of histidine decarboxylase by rhodanines

The inhibition of rat gastric histidine decarboxylase in vitro by rhodanine and 26 of its 3- or 5-substituted derivatives has been investigated. Inhibitory activities of the 3-substituted derivatives of rhodanine ranged from i 50 values of 2 × 10 −6 M to 2 × 10 −4 M. Eight 5-substituted derivatives were relatively inactive, yielding i 50 values higher than 5 × 10 −4 M. Studies performed with 3- p- chlorobenzylrhodanine ( i 50 = 3 × 10 −6 M) on preparations of histidine decarboxylase holoenzyme and apoenzyme indicated that only the holoenzyme form of the decarboxylase is susceptible to inhibition on treatment with an excess of the inhibitor, and that this inhibition is irreversible. The results are consistent with a mechanism of inhibition that involves a condensation reaction between the 5-methylene carbon of the rhodanine ring and enzyme-bound pyridoxal phosphate.

Physical-Chemical Studies on the Pyridoxal Phosphate Binding Site in Sodium Borohydride-reduced and Native Phosphorylase

Abstract NaBH4-reduced phosphorylase b is a catalytically active species which retains many of the properties of the native enzyme. One property that is strikingly different, however, is the spectral behavior of bound pyridoxal phosphate. At pH 7.0, the pyridoxal phosphate shows essentially no absorption at 333 nm, where pyridoxal phsophate in the native enzyme absorbs; absorption at 333 nm returns upon lowering the pH. The 333 nm band at low pH in the reduced enzyme was found to be optically active, (ΔA/A) 333 nm ≅ 0.7 x 10-3. Circular dichroism of the reduced phosphorylase b at pH 7.0 indicated that no new band was formed above 300 nm; the ultraviolet spectrum also provided no evidence for a band above 300 nm. Guanidine·HCl (4.25 m) and sodium dodecyl sulfate (0.3%) caused the appearance of a band near 330 nm at pH 6.8. Difference spectra of reduced phosphorylase b (±sodium dodecyl sulfate; pH 5 versus pH 7) indicated that the hidden band must be below 300 nm. AMP (0.01 m) was shown to cause partial reversal of the hidden band to 333 nm conversion at pH 6.0; ATP was also effective. Substrates and AMP had no effect on the spectrum of the reduced phosphorylase b at pH 6.8. Ultracentrifugal studies showed that native and NaBH4-reduced enzyme have different conformations. The data were interpreted to mean that the 333 nm and hidden band forms of NaBH4-reduced phosphorylase represent different enzyme conformations. The hidden band form is thought to represent a form of pyridoxal phosphate in which the pyridinium nitrogen is unprotonated and the 3-hydroxyl group undissociated or strongly hydrogen bonded; the 333 nm species is believed to be a dipolar form of pyridoxal phosphate which is exposed to solvent. Spectral properties of enzyme-bound pyridoxal phosphate in native phosphorylase were compared to NaBH4-reduced enzyme, and it was suggested that pyridoxal phosphate exists in these two forms at pH 7.0 in the same microscopic environment. Circular dichroism of native phosphorylase b at 422 nm was observed. The presence of phosphate (0.01 m) and AMP (0.001 m) did not perturb the observed long wave length circular dichroism. Solvent perturbation studies showed that enzyme-bound pyridoxal phosphate in native phosphorylase is not available to ethylene glycol. From our results, evaluation of model compound data, and fluorescence results, a new structure was proposed for the 333 nm form for native phosphorylase. This species is thought to be a neutral tautomeric imine of pyridoxal phosphate.

Imidazolylacetolphosphate: l-Glutamate aminotransferase-mechanism of action

Some kinetic, chemical, and spectrographic properties of imidazolylacetolphosphate: l glutamate aminotransferase have been examined. The data support a shuttle mechanism for the transamination reaction involving conversion of the enzyme-bound pyridoxal phosphate to pyridoxamine phosphate.

Reaction of dithionite and bisulfite with pyridoxal and pyridoxal enzymes

Both bisulfite and dithionite produce marked changes in the spectra of pyridoxal, pyridoxal phosphate, and pyridoxal phosphate enzymes. The spectrum of pyridoxal phosphate in bisulfite solutions resembles that of the addition product of pyridoxal phosphate with cysteine. The spectral changes produced by dithionite are distinctly different from those produced by bisulfite and, unlike the latter, are not prevented by acetone. As judged by spectral change, pyridoxal phosphate enzymes appear less reactive with bisulfite than with dithionite, relative to the reactivity of the free coenzyme. The nature of the dithionite reaction and of its products has not been determined. However, the difference spectrum attributable to dithionite reaction with enzyme-bound pyridoxal phosphate, while distinguishable from that of a flavin difference spectrum must be kept in mind in interpreting presumptive flavin difference spectra. In addition, dithionite may be a useful reagent for delineating, by difference spectra, the characteristic low 400 mμ peak of pyridoxal phosphate enzymes.

Reactivating effect of some naturally occurring dithiol compounds on threonine deaminase

The biodegradative threonine deaminases have been purified to homogeneity and crystallized from the extracts of Escherichia coli (E. coli) and Clostridium tetranomorphum, respectively. It has been demonstrated that deaminases are composed of four identical subunits, each monomer containing one mole of pyridoxal phosphate. The deamination reaction mechanism studied using the E. coli enzyme is compatible with the mechanism derived from the pyridoxal-catalyzed α,β-elimination reaction in the non-enzymic model system, although direct evidence for the participation of pyridoxal phosphate in the enzyme reaction is lacking. Spectral and circular dichroic changes of the enzyme-bound pyridoxal phosphate after addition of the substrate, L-threonine, were postulated to represent the formation of a complex between pyridoxal phosphate and the dehydrated reaction intermediate. Optical studies indicate that optical changes occur prior to the β-elimination reaction. Kinetic analyses have indicated that the enzyme exists as an inactive form under the conditions of these optical experiments. These phenomena could be interpreted as indicating that the conversion of this inactive enzyme into an active form occurs after addition of L-threonine. These optical changes are observed during the reaction and this conversion step appears to be the rate-limiting step of the overall reaction under the conditions employed.

Circular dichroism of biodegradative threonine deaminase

Biodegradative threonine deaminase of Escherichia coli ( Umbarger and Brown, 1957) contains pyridoxal phosphate (PLP) as a cofactor and is activated by AMP ( Wood and Gunsalus, 1949; Phillips and Wood, 1964; Hirata et al. , 1965 ). In addition to the catalytic function through Schiff base formation with substrate, the enzymebound PLP was shown to contribute to the stabilization of the enzyme conformation ( Tokushige, 1967). In this paper is described the circular dichroism (CD) of PLP in the biodegradative threonine deaminase in relation to the catalytic reaction; the results are pertinent to the mode of binding of PLP to the enzyme protein, and their interaction.