Pyridoxal Phosphate Binding Site Research Articles

Localization of pyrodoxal phosphate binding site on the mero-receptor domain of the glucocorticoid receptor

Previous studies have demonstrated that the vitamin pyridoxal phosphate can alter the physicochemical properties of glucocorticoid receptors. We now report the localization of a pyridoxal phosphate binding site within the mero-receptor domain of this glucocorticoid receptor. Mero-glucocorticoid receptors that are generated by trypsin (10 μg/ml) or chymotrypsin (100 μg/ml) digestion of intact receptors sediment as 2.6 S species on 5–20% sucrose gradients in the presence or absence of pyridoxal phosphate. Mero-glucocoritcoid receptors prepared by exogenous proteinases are hydrophobic and show no affinity for DEAE Bio-Gel A. Treating either trypsin-generated or chymotrypsin-generated mero-receptors with pyridoxal phosphate rapidly converts the proteins (60 and 35%, respectively) into forms that bind to DEAE Bio-Gel A. Induction of DEAE binding is specific to pyridoxal phosphate, for treating mero-receptors with pyridoxal, pyridoxamine or pyridoxine phosphate is ineffective. Furthermore, DEAE binding cannot be induced by adding other pyridoxal phosphate-treated cytosols to untreated mero-receptors. High-resolution polyacrylamide gel isoelectric focussing studies indicated that treating mero-receptor generated by either proteinase with pyridoxal phosphate shifted the isoelectric points of lower pH values. The conversion of the mero-receptor to a more acidic form also occurred when the intact glucocorticoid receptor was treated with the vitamin prior to proteolysis. These studies localize at least one pyridoxal phosphate binding site on the mero-receptor domain of the rat thymocyte glucocorticoid receptor.

Pyridoxal phosphate modified cytochromes c. Identification and electron transfer properties

The preparation, purification and characterization of the three singly, three doubly and one triply substituted derivatives of cytochrome c modified by pyridoxal phosphate (PLP) at lysine residues are reported. The PLP positions in PLP derivatives were determined by the amino acid analysis and sequence of PLP peptides. The results identified the lysine at position 86 in one of the singly substituted, lysine 79 in the other singly substituted and lysines 86 and 79 in the third doubly substituted cytochrome c derivatives. The area surrounding phenylalanine 82 forms the predominant PLP binding site on the cytochrome c molecule. The visible, CD and proton NMR spectra, the full intensity of the conformation-sensitive 695 nm band and the oxidation-reduction properties provide evidence to confirm the conclusion that singly and doubly substituted PLP cytochromes c retain the native conformation. The ability to restore both succinate and ascorbate/TMPD oxidation in cytochrome c-depleted mitochondria decreases in the order: native cytochrome c > PLP-Lys-79-cytochrome c > PLP-Lys-86-cytochrome c > PLP-Lys-79,86-cytochrome c > triply substituted derivative.

10] Pyridoxal phosphate-binding site in enzymes: Reduction and comparison of sequences

Publisher Summary This chapter illustrates pyridoxal phosphate-binding site in enzymes. In pyridoxal phosphate-dependent enzymes, the formyl group at position 4 of pyridoxal-P forms an aldimine bond with the ɛ -amino group of a specific lysyl residue. Involvement of a single particular lysyl residue indicates that there is a specific domain structure favorable for stabilizing the aldimine bond which, otherwise, is readily hydrolyzed. X-Ray analyses of both cytosolic and mitochondrial isoenzymes of aspartate aminotransferase, representative pyridoxal-P-dependent enzymes, provide details of interaction between pyridoxal-P and the enzyme side chains in the coenzyme-accommodating active site. Only the Re side of the aldimine bond is open to solvent, the Si face being shielded from solvent. This stereospecific status of the bound coenzyme has been remarkably identical among a variety of pyridoxal-P-dependent enzymes. At neutral pH, pyridoxal-P absorbs at 390 nm, which shifts more or less to a longer wavelength (410-430 nm) upon the formation of an aldimine linkage with amino compounds or apoenzymes. Pyridoxal-P-aldimine bond is reduced by borohydride or borocyanohydride under mild conditions. The reduction renders the aldimine bond highly stable to hydrolysis. The resulting reduced enzyme is fragmented by either proteolytic or chemical cleavage, followed by isolation of a phosphopyridoxyl peptide and its structural determination.

Phosphorus 31 nuclear magnetic resonance study of tryptophanase. Pyridoxal phosphate-binding site.

The pyridoxal phosphate-dependent enzyme tryptophanase has been investigated using 31P nuclear magnetic resonance at 72.86 MHz. In the native enzyme, the pyridoxal-P 31P chemical shift was found to be 3.55 ppm and independent of pH, indicating that the dianionic phosphate group of the cofactor is not accessible to solvent. Binding of the competitive inhibitor, beta-phenyl-DL-serine, results in the formation of the transaldimination complex. This complex is fixed to the enzyme via the dianionic phosphate group of the cofactor: again the observed shift is independent of pH. In both cases, restricted rotational freedom of the phosphate group around the C-O bond linking the phosphate ester to the pyridine moiety of the cofactor could be asserted from line width data. Addition of the competitive inhibitor, L-alanine, to tryptophanase produces the quinonoid intermediate. The phosphate group of this complex has lost its specific interaction (probably a salt bridge) with the protein, as indicated by the pH dependence of the chemical shift.

Essential arginine residues at the pyridoxal phosphate binding site of brain γ-aminobutyrate aminotransferase

Summary Mouse brain γ-aminobutyrate aminotransferase was inactivated by phenylglyoxal in a time and concentration dependent manner. Pyridoxal phosphate but not γ-aminobutyric acid or α-ketoglutarate afforded some protection against the drug-induced inactivation. [14C]Phenylglyoxal was incorporated into the enzyme at a similar rate to that of the inactivation. Subsequent hydrolysis of the enzyme demonstrated the presence of a [14] phenylglyoxal-arginine complex. The phenylglyoxal is probably modifying an arginine residue at or near the pyridoxal phosphate binding site of γ-aminobutyrate aminotransferase.

Kinetics of the inactivation of Escherichia coli glutamate apodecarboxylase by phenylglyoxal

The possible interaction of the phosphate moiety of pyridoxal phosphate with a guanidinium group in glutamate apodecarboxylase was investigated. The holoenzyme is not inactivated significantly by incubation with butanedione, glyoxal, methylglyoxal, or phenylglyoxal. However, the apoenzyme is inactivated by these arginine reagents in time-dependent processes. Phenylgloxal inactivates the apoenzyme most rapidly. The inactivation follows pseudo-first-order kinetics at high phenylglyoxal to apoenzyme ratios. The rate of inactivation is proportional to phenylglyoxal concentration, increases with increasing pH, and is also dependent on the type of buffer present. The rate of inactivation of the apoenzyme by phenylglyoxal is fastest in bicarbonate — carbonate buffer and increases with increasing bicarbonate — carbonate concentration. Phosphate, which inhibits the binding of pyridoxal phosphate to the apoenzyme, protects the apodecarboxylase against inactivation by phenylglyoxal. When the apodecarboxylase is inactivated with [ 14C]phenylglyoxal, approximately 1.6 mol of [ 14C]phenylglyoxal is incorporated per mol subunit. The phenylglyoxal is thought to modify an arginyl residue at or near the pyridoxal phosphate binding site of glutamate apodecarboxylase.

Isolation of two different molecular weight polypeptides copurifying with rat liver tyrosine aminotransferase

An affinity chromotography resin highly specific for rat liver tyrosine aminotransferase (EC 2.6.1.5) has been synthesized and used in the purification of this enzyme. The structure of the resin, N-(5′-phosphopyridoxyl)- l-tyrosyl-aminoocytl-Sepharose 4B, was designed to resemble the tyrosine-pyridoxal phosphate Schiff's base intermediate in the reaction pathway catalyzed by this enzyme. Use of this resin in combination with octyl-agarose chromatography on partially purified enzyme resulted in a tyrosine aminotransferase preparation with a specific activity of about 450 units/mg protein. When analyzed on one-dimensional polyacrylamide-sodium dodecyl sulfate slab gels, the highly purified enzyme was composed of two polypeptides with molecular weights of about 56,000 and 53,000. Radioiodinated tryptic peptides from each of these polypeptides were essentially identical following two-dimensional analysis. Although the two polypeptides could not be separated from each other in an active form, it was found that (i) both polypeptides have pyridoxal phosphate-binding sites, (ii) the coenzyme is probably bound to both polypeptides as a Schiff's base, (iii) both polypeptides have binding sites for l-tyrosine and l-glutamic acid, the two specific substrates for the enzyme, and (iv) both polypeptides can catalyze the formation of the initial amino acid-pyridoxal phosphate Schiff's base adduct in the overall reaction pathway. Since the ratios of these polypeptides differed from preparation to preparation of purified enzyme, the 53,000 M r species probably arises by proteolysis of tyrosine aminotransferase in crude liver extracts. These results imply that if tyrosine aminotransferase isozymes exist, they are not the result of translation products produced by different structural genes.

Selective proteolysis of the β2 subunit of Serratia marcescens tryptophan synthase

Mild digestion of Serratia marcescens tryptophan synthase β 2 subunit produces a modified β 2 subunit (nicked β 2). The nicked β 2 subunit remains essentially intact and is immunochemically reactive with native β 2 subunit antiserum. Denaturation of the nicked β 2 subunit yields two principal peptide fragments whose minimum molecular weights are 29,500 and 13,400. Loss of enzyme activity is associated with the selective proteolysis. The enzyme cofactor pyridoxal phosphate binding site is on the larger fragment. Following separation of the fragments by urea-gel chromatography, the separated peptides retain immunological cross-reactivity with native β 2 subunit antiserum. These fragments apparently represent two domains that comprise the native Holo β 2 subunit. The immunochemical data suggest that these fragments, when isolated, can assume some tertiary structure and that they may exist as such prior to β monomer or β 2 dimer assembly. The folded fragments may represent intermediates in the biosynthesis of the β 2 subunit as has been suggested for the E. coli enzyme ( A. Högberg-Raibaud and M. E. Goldberg, 1977, Proc. Nat. Acad. Sci. USA 74, 442; Biochemistry 16, 4014).

Amino‐Acid Sequence of the Pyridoxal‐Phosphate‐Binding Site in Escherichia coli Maltodextrin Phosphorylase

The primary sequence of the pyridoxal 5′‐phosphate binding site in Escherichia coli maltodextrin phosphorylase was determined as part of a study on the role of pyridoxal phosphate for structure and function of phylogenetically different phosphorylases with different regulatory properties. Maltodextrin phosphorylase was isolated and purified by an improved procedure. The enzyme contains 2 mol pyridoxal phosphate per mol of dimer in accordance with other phosphorylases. The sodiumborohydride‐reduced phosphorylase was cleaved by Staphyllococcus aureus protease and a peptide carrying the fluorescent pyridoxyl marker was isolated by gel filtration and differential ion‐exchange chromatography before and after phosphatase treatment. A sequence, 21 residues long, was established partially by automatic Edman degradation and from overlap of proteolytic fragments as: Ala‐Ser‐Gly‐Thr‐Gly‐Asn‐Met‐Lys(PPxy)‐Leu‐Ala‐Leu‐Asp‐Gly‐Ala‐Leu‐Thr‐Val‐Gly‐Thr‐Leu‐Asp. The part of the sequence preceding Lys(PPxy), N6‐(phosphopyridoxyl)‐l‐lysine, is identical with the corresponding sequence in rabbit muscle phosphorylase. Among the five amino acid exchanges observed distal to the cofactor binding site, four can be ascribed to conservative substitutions. A comparison of all known sequences of pyridoxal phosphate binding sites in phosphorylases shows a region of high mutability among the four amino acid residues immediately following the cofactor binding site.

Biosynthesis of bacterial glycogen. Incorporation of pyridoxal phosphate into the allosteric activator site and an ADP-glucose-protected pyridoxal phosphate binding site of Escherichia coli B ADP-glucose synthase.

[3H]Pyridoxal-P can be covalently incorporated into Escherichia coli B mutant strain AC70R1 ADP-glucose synthase by reduction with NaBH4. Two distinct lysine residues can be modified by the allosteric activator pyridoxal-P. Incorporation of [3H]pyridoxal-P in the presence of substrate ADP-glucose + MgCl2 prevents pyridoxylation of an ADP-glucose-protected site and allows modification of the allosteric activator site. Incorporation of [3H]pyridoxal-P in the presence of allosteric effectors fructose-P2, 5'-AMP, or hexanediol-1,6-P2, protects against pyridoxylation of the allosteric activator site, and allows modification of the ADP-glucose-protected site. Incorporation of pyridoxal-P into the allosteric activator site results in modified enzyme of high activity form, even in the absence of fructose-P2. This modified enzyme, when assayed in the absence of fructose-P2, exhibits activation kinetics similar to nonpyridoxylated enzyme assayed in the presence of fructose-P2 and is still inhibited by 5'-AMP. These data suggest that the allosteric activator site of pyridoxylation is the fructose-P2 binding site, and is distinct from the inhibitor 5'-AMP binding site. Incorporation of pyridoxal-P into the ADP-glucose-protected site results in a decrease in enzyme activity. This pyridoxylated lysine could be involved with the binding of thesubstrates ADP-glucose, alpha-glucose-1-P, or PPi, or participate in the catalytic mechanism of the enzyme.

Affinity labeling of the pyridoxal phosphate binding site of the beta2 subunit of Escherichia coli tryptophan synthase

We have synthesized bromoacetylpyridoxamine phosphate and bromoacetylpyridoxamine and have shown that they meet three criteria for affinity labels of the beta2 subunit of tryptophan synthase: (i) the kinetic data of inactivation indicate that a binary complex is formed prior to covalent attachment; (ii) inactivation is largely prevented by the presence of pyridoxal phosphate; and (iii) inactivation is stoichiometric with incorporation of 0.7 to 0.8 mol of chromophore/mol of beta monomer. Our conclusion that inactivation of the apo beta2 subunit by bromoacetylpyridoxamine phosphate is due to the modification of cysteine is based on the disappearance of 1 mol of -SH/beta monomer and on the finding that [14C]carboxymethyl derivative in the acid hydrolysate of the protein modified by bromo[14C]acetylpyridixamine phosphate. A 39-residue tryptic peptide containing this essential cysteine has been isolated and purified from the bromo[14C]acetylpyridoxamine phosphate-labeled beta2 subunit.

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

Our findings that the apo β 2 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 β 2 subunit labeled selectively at SH-I or SH-II show that both sulfhydryl residues are located in the F 1 fragment which also contains the pyridoxal phosphate binding site.

Threonine deaminase from a nonsense mutant of Escherichia coli requiring isoleucine or pyridoxine: evidence for half-of-the-sites reactivity.

The mutant IP7 of Escherichia coli B requires isoleucine or pyridoxine for growth as a consequence of a mutation in the gene coding for biosynthetic threonine deaminase. The mutation of IP7 was shown to be of the nonsense type by the following data: (1) reversion to isoleucine prototrophy involves the formation of external suppression at a high frequency, as shown by transduction experiments; and (ii) the isoleucine requirement is suppressed by lysogenization with a phage carrying the amber suppressor su-3. Cell extracts of the mutant strain contain a low activity of threonine deaminase. The possibility that this activity is biodegradative was ruled out by kinetic experiments. The mutant threonine deaminase was purified to homogeneity by conventional procedures. The enzyme is a dimer of identical subunits of an approximate molecular weight of 43,000 (Grimminger and Feldner, 1974), whereas the wild-type enzyme is a tetramer of 50,000-dalton subunits (Calhoun et al., 1973; Grimminger et al., 1973). The mutant enzyme is not inhibited by isoleucine and does not bind isoleucine, as shown by equilibrium dialysis experiments. Pyridoxal phosphate enhances the maximum catalytic activity of the mutant enzyme by a factor of five, whereas the wild-type enzyme is not affected. In wild-type and mutant threonine deaminase the ratio of protein subunits and bound pyridoxal phosphate is 2:1. The activation of threonine deaminase from strain IP7 is due to a second coenzyme binding site, as shown by (i) spectrophotometric titration of the enzyme with pyridoxal phosphate and by (ii) measurement the pyridoxal phosphate content of the enzyme after sodium borohydride reduction of the protein. The observation of one pyridoxal phosphate binding site per peptide dimer in the wild-type enzyme and of two binding sites per dimer in the mutant strongly suggests that one of the potential sites in the wild-type enzyme is masked by allosteric effects. The factors responsible for the half-of-the-sites reactivity of the coenzyme sites appear to be nonoperative in the mutant protein.

A comparison of intact human red blood cells and resealed and leaky ghosts with respect to their interactions with surface labelling agents and proteolytic enzymes

Resealed ghosts and intact red blood cells were directly compared with respect to their interactions with surface probes and to digestion by pronase. The amount and pattern of labelling of surface proteins by 4.4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) and by pyridoxal phosphate-borohydride (as seen after sodium dodecylsulfate/acrylamide gel electrophoresis) was substantially the same in cells and resealed ghosts under conditions in which a relatively small change would be apparent. In each membrane system, DIDS labels a protein component of apparent molecular weight 95 000 and pyridoxal phosphate labels the same protein plus three glycoprotein components. The sensitivity of surface proteins and of DIDS and pyridoxal phosphate-labelled sites to pronase was also similar in the cells and resealed ghosts. The glycoproteins were digested, in each case, and the 95 000 (molecular weight) protein was largely split into two portions of apparent molecular weights 65 000 and 35 000, with both portions containing DIDS and pyridoxal phosphate binding sites. The pattern of labelling of “leaky” ghosts by pyridoxal phosphate in the presence of hemoglobin was similar to the labelling of intact cells, provided that the pyridoxal phosphate was present on both the outside and inside of the cells. Virtually all of the major protein components visible by staining on acrylamide gels were labelled. It is concluded that none of the probes could detect any substatial differences in reactivity of proteins of the outer surface of the membrane of the ghosts as compared to the cells and that no irreversible changes in membrane protein conformation or arrangement occur as a consequence of lysis and resealing of ghosts, that are detectable by the reported procedures.

The pyridoxal phosphate-binding site of rabbit muscle aldolase

Under appropriate conditions pyridoxal phosphate forms a Schiff-base derivative with a specific lysine residue in rabbit muscle aldolase, with the incorporation of slightly less than 1 equiv of pyridoxal phosphate per enzyme subunit. Reduction of the Schiff base with tritium-labeled borohydride introduces a radioactive label at this site. A tryptic peptide containing the labeled lysine residue has been isolated and found to possess the following sequence: Gly-Gly-Val-Val-Gly-Ile-Lys ∗-Val-Asp-Lys, where the asterisk indicates the modified lysine residue.

The thiol groups of rat liver cystathionase. Influence of pyridoxal phosphate, L-homoserine and L-alanine on the effect of p-chloromercuribenzoate and 5,5-dithiobis-(2-nitrobenzoate) on the enzyme.

Rat liver cystathionase (homoserine hydrolyase) catalyzes the deamination of homoserine and the desulfhydration of cysteine. A study has been made of the SH groups of the highly purified enzyme. The titration of the SH groups by p‐chloromercuribenzoate and 5,5′‐dithiobis‐(2‐nitrobenzoic acid) indicated that the enzyme contains approximatively 12 SH per mole (mol. wt. 170000) of native enzyme and 20 SH per mole of urea‐denaturated enzyme.On the basis of their reactivity towards SH reagents the SH groups of cystathionase have been classified into 4 classes.Addition of pyridoxal phosphate, the coenzyme of cystathionase, prior to treatment with SH reagents did not modify the availability of SH groups for SH reagents; however, addition of homoserine, a substrate for cystathionase, or of l‐alanine, a competitive inhibitor for both homoserine deamination and cysteine desulfhydration, led to a striking protection of several SH groups, 4 and 8, respectively.The over‐all results are discussed in relation to the hypothesis of a “double” active center composed of one site necessary to the binding of homoserine and one other involved in the binding of cysteine. In addition the hypothesis that cystathionase may consist of subunits is considered, taking into account the symmetry of the SH groups in each class and the number of pyridoxal phosphate binding sites on the protein.

Interaction of Pyridoxal Analogues with Glutamate Apodecarboxylase and Aspartate Apoaminotransferase

The interaction of a variety of pyridoxal analogues with glutamate decarboxylase and aspartate aminotransferase has been studied. Pyridoxal phosphate N-oxide, N-methylpyridoxal phosphate, 3-O-methylpyridoxal phosphate, 4-de-(formyl)-4-vinyl pyridoxal phosphate, and pyridoxal analogues which contain modified substituents in position 5 (CH2CH2OPO3H2, CH(CH3)OPO3H2, CH2PO3H2, and CH2-CH2COOH) are bound at the pyridoxal phosphate binding site of the enzymes. The reaction of the analogues with the apoenzymes prevented activation with pyridoxal phosphate. Pyridoxal phosphate, pyridoxal phosphate N-oxide, O-methylpyridoxal phosphate, and the homologue of pyridoxal phosphate bind to apodecarboxylase to give absorption spectra characteristic of protonated Schiff bases. The rates of binding, the coenzyme constants, and the activation of the enzymes were determined for the analogues with aspartate apoaminotransferase and glutamate apodecarboxylase. The phosphate group of the coenzyme plays an important role in binding to the enzymes and in maintaining the enzymes and coenzymes in the proper conformation for catalysis. There are more stringent structural requirements for interaction of pyridoxal phosphate with the decarboxylase than with the transaminase. The free phenolic hydroxyl group and the free pyridine ring nitrogen are of importance in the interaction of pyridoxal phosphate with glutamate apodecarboxylase, but not as much as with aspartate apoaminotransferase. However, the phenolic hydroxyl group and the pyridine ring nitrogen are very important in the activation of the transminase. The addition of pyridoxal phosphate N-oxide to apodecarboxylase yields enzyme which is 29% as active as the native holoenzyme. The decarboxylase-N-oxide complex and the native glutamate decarboxylase have similar Km values for glutamate and Ki values for glutarate.

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.

Active site peptides from glycogen phosphorylase

The isolation and sequence of active site peptides from glycogen phosphorylase b is described. These cysteine peptides become exposed to preferential labeling upon removal of pyridoxal 5′-phosphate (PLP) from the enzyme and they have the sequence Ala-Cys, Asx-Ala-Cys-Asp and Asx-Glx-Lys-Cys-Gly-Gly. The first two of these peptides appear to be derived from the PLP binding site of phosphorylase.

Primary structure of pyridoxal phosphate binding site in the mitochondrial and extramitochondrial aspartate aminotransferases from pig heart muscle. Chymotryptic peptides.

Primary structure of pyridoxal phosphate binding site in the mitochondrial and extramitochondrial aspartate aminotransferases from pig heart muscle. Chymotryptic peptides.