2-Amino-3-ketobutyrate CoA ligase of Escherichia coli: stoichiometry of pyridoxal phosphate binding and location of the pyridoxyllysine peptide in the primary structure of the enzyme

Pure 2-amino-3-ketobutyrate CoA ligase from Escherichia coli, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the L-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, is a dimeric enzyme (Mr = 84,000) that requires pyridoxal 5'-phosphate as coenzyme for catalytic activity. Reduction of the hololigase with tritiated NaBH4 yields an inactive, radioactive enzyme adduct; acid hydrolysis of this adduct allowed for the isolation and identification of epsilon-N-pyridoxyllysine. Quantitative determinations established that 2 mol of pyridoxal 5'-phosphate are bound per mol of dimeric enzyme. After the inactive, tritiated enzyme adduct was digested with trypsin, a single radioactive peptide containing 23 amino acids was isolated and found to have the following primary structure: Val-Asp-Ile-Ile-Thr-Gly-Thr-Leu-Gly-Lys*-Ala-Leu-Gly-Gly-Ala-Ser-Gly-Gly -Tyr-Thr-Ala-Ala-Arg (where * = the lysine residue in azomethine linkage with pyridoxal 5'-phosphate). This peptide corresponds to residues 235-257 in the intact protein; 10 residues around the lysine residue have a high level of homology with a segment of the primary structure of 5-aminolevulinate synthase from chicken liver.     

Circular permutation of 5-aminolevulinate synthase. Mapping the polypeptide chain to its function

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and some prokaryotes. The enzyme functions as a homodimer and requires pyridoxal 5'-phosphate as a cofactor. Although the roles of defined amino acids in the active site and catalytic mechanism have been recently explored using site-directed mutagenesis, much less is known about the role of the 5-aminolevulinate synthase polypeptide chain arrangement in folding, structure, and ultimately, function. To assess the importance of the continuity of the polypeptide chain, circularly permuted 5-aminolevulinate synthase variants were constructed through either rational design or screening of an engineered random library. One percent of the random library clones were active, and a total of 21 active variants had sequences different from that of the wild type 5-aminolevulinate synthase. Out of these 21 variants, 9 displayed unique circular permutations of the 5-aminolevulinate synthase polypeptide chain. The new termini of the active variants disrupted secondary structure elements and loop regions and fell in 100 amino acid regions from each terminus. This indicates that the natural continuity of the 5-aminolevulinate synthase polypeptide chain and the sequential arrangement of the secondary structure elements are not requirements for proper folding, binding of the cofactor, or assembly of the two subunits. Furthermore, the order of two identified functional elements (i.e. the catalytic and the glycine-binding domains) is apparently irrelevant for proper functioning of the enzyme. Although the wild type 5-aminolevulinate synthase and the circularly permuted variants appear to have similar, predicted overall tertiary structures, they exhibit differences in the arrangement of the secondary structure elements and in the cofactor-binding site environment. Taken together, the data lead us to propose that the 5-aminolevulinate synthase overall structure can be reached through multiple or alternative folding pathways.     

Mechanism of 5-aminolevulinate synthase and the role of the protein environment in controlling the cofactor chemistry.

5-Aminolevulinate synthase, a pyridoxal 5'-phosphate-dependent enzyme of the alpha-oxoamine synthase family, catalyzes the first step of the heme biosynthetic pathway in mammalian cells. This reaction entails the condensation of glycine with succinyl-coenzyme A to yield 5-aminolevulinate, carbon dioxide and CoA. Mutations in the erythroid aminolevulinate synthase gene lead to a defective enzyme and are associated with the erythropoietic disorder X-linked sideroblastic anemia. In the past few years, rapid scanning-stopped-flow spectroscopy and chemical quenched-flow studies of the ALAS reaction, under single- and multi-turnover conditions, have provided important results for the interpretation of the catalytic mechanism. In particular, the role of the protein scaffold in modulating the chemical reactivity of the pyridoxal 5'-phosphate cofactor and, thus, the catalytic pathway of ALAS has been investigated in our laboratory using transient kinetics and global analysis of the kinetic data.     

5-Aminolevulinate synthase and the first step of heme biosynthesis

5-Aminolevulinate synthase catalyzes the condensation of glycine and succinyl-CoA to yield 5-aminolevulinate. In animals, fungi, and some bacteria, 5-aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway. Mutations on the human erythroid 5-aminolevulinate synthase, which is localized on the X-chromosome, have been associated with X-linked sideroblastic anemia. Recent biochemical and molecular biological developments provide important insights into the structure and function of this enzyme. In animals, two aminolevulinate synthase genes, one housekeeping and one erythroid-specific, have been identified. In addition, the isolation of 5-aminolevulinate synthase genomic and cDNA clones have permitted the development of expression systems, which have tremendously increased the yields of purified enzyme, facilitating structural and functional studies. A lysine residue has been identified as the residue involved in the Schiff base linkage of the pyridoxal 5-phosphate cofactor, and the catalytic domain has been assigned to the C-terminus of the enzyme. A conserved glycine-rich motif, common to all aminolevulinate synthases, has been proposed to be at the pyridoxal 5phosphate-binding site. A heme-regulatory motif, present in the presequences of 5-aminolevulinate synthase precursors, has been shown to mediate the inhibition of the mitochondrial import of the precursor proteins in the presence of heme. Finally, the regulatory mechanisms, exerted by an iron-responsive element binding protein, during the translation of erythroid 5-aminolevulinate synthase mRNA, are discussed in relation to heme biosynthesis.     

Lysine-313 of 5-aminolevulinate synthase acts as a general base during formation of the quinonoid reaction intermediates

5-Aminolevulinate synthase catalyzes the condensation of glycine and succinyl-CoA to form CoA, carbon dioxide, and 5-aminolevulinate. This represents the first committed step of heme biosynthesis in animals and some bacteria. Lysine 313 (K313) of mature murine erythroid 5-aminolevulinate synthase forms a Schiff base linkage to the pyridoxal 5'-phosphate cofactor. In the presence of glycine and succinyl-CoA, a quinonoid intermediate absorption is transiently observed in the visible spectrum of purified murine erythroid ALAS. Mutant enzymes with K313 replaced by glycine, histidine, or arginine exhibit no spectral evidence of quinonoid intermediate formation in the presence of glycine and succinyl-CoA. The wild-type 5-aminolevulinate synthase additionally forms a stable quinonoid intermediate in the presence of the product, 5-aminolevulinate. Only conservative mutation of K313 to histidine or arginine produces a variant that forms a quinonoid intermediate with 5-aminolevulinate. The quinonoid intermediate absorption of these mutants is markedly less than that of the wild-type enzyme, however. Whereas the wild-type enzyme catalyzes loss of tritium from [2-3H2]-glycine, mutation of K313 to glycine results in loss of this activity. Titration of the quinonoid intermediate formed upon binding of 5-aminolevulinate to the wild-type enzyme indicated that the quinonoid intermediate forms by transfer of a single proton with a pK of 8.1 +/- 0.1. Conservative mutation of K313 to histidine raises this value to 8.6 +/- 0.1. We propose that K313 acts as a general base catalyst to effect quinonoid intermediate formation during the 5-aminolevulinate synthase catalytic cycle.     

Pre-steady-state reaction of 5-aminolevulinate synthase. Evidence for a rate-determining product release

5-Aminolevulinate synthase (ALAS) is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and the alpha-subclass of purple bacteria. The pyridoxal 5'-phosphate cofactor at the active site undergoes changes in absorptive properties during substrate binding and catalysis that have allowed us to study the kinetics of these reactions spectroscopically. Rapid scanning stopped-flow experiments of murine erythroid 5-aminolevulinate synthase demonstrate that reaction with glycine plus succinyl-CoA results in a pre-steady-state burst of quinonoid intermediate formation. Thus, a step following binding of substrates and initial quinonoid intermediate formation is rate-determining. The steady-state spectrum of the enzyme is similar to that formed in the presence of 5-aminolevulinate, suggesting that release of this product limits the overall rate. Reaction of either glycine or 5-aminolevulinate with ALAS is slow (kf = 0.15 s-1) and approximates kcat. The rate constant for reaction with glycine is increased at least 90-fold in the presence of succinyl-CoA and most likely represents a slow conformational change of the enzyme that is accelerated by succinyl-CoA. The slow rate of reaction of 5-aminolevulinate with ALAS is 5-aminolevulinate-independent, suggesting that it also represents a slow isomerization of the enzyme. Reaction of succinyl-CoA with the enzyme-glycine complex to form a quinonoid intermediate is a biphasic process and may be irreversible. Taken together, the data suggest that turnover is limited by release of 5-aminolevulinate or a conformational change associated with 5-aminolevulinate release.     

The role of tyrosine 121 in cofactor binding of 5-aminolevulinate synthase.

5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthesis in nonplant eukaryotes and some prokaryotes. It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor. Tyr-121 is a conserved residue in all known sequences of 5-aminolevulinate synthases. Further, it corresponds to Tyr-70 of Escherichia coli aspartate aminotransferase, which has been shown to interact with the cofactor and prevent the dissociation of the cofactor from the enzyme. To test whether Tyr-121 is involved in cofactor binding in murine erythroid 5-aminolevulinate synthase, Tyr-121 of murine erythroid 5-aminolevulinate synthase was substituted by Phe and His using site-directed mutagenesis. The Y121F mutant retained 36% of the wild-type activity and the Km value for substrate glycine increased 34-fold, while the activity of the Y121H mutant decreased to 5% of the wild-type activity and the Km value for glycine increased fivefold. The pKa1 values in the pH-activity profiles of the wild-type and mutant enzymes were 6.41, 6.54, and 6.65 for wild-type, Y121F, and Y121H, respectively. The UV-visible and CD spectra of Y121F and Y121H mutants were similar to those of the wild-type with the exception of an absorption maximum shift (420 --> 395 nm) for the Y121F mutant in the visible spectrum region, suggesting that the cofactor binds the Y121F mutant enzyme in a more unrestrained manner. Y121F and Y121H mutant enzymes also exhibited lower affinity than the wild-type for the cofactor, reflected in the Kd values for pyridoxal 5'-phosphate (26.5, 6.75, and 1.78 microM for Y121F, Y121H, and the wild-type, respectively). Further, Y121F and Y121H proved less thermostable than the wild type. Taken together, these findings indicate that Tyr-121 plays a critical role in cofactor binding of murine erythroid 5-aminolevulinate synthase.     

5-Enolpyruvyl shikimate 3-phosphate synthase from Escherichia coli. Identification of Lys-22 as a potential active site residue

5-Enolpyruvyl shikimate 3-phosphate synthase catalyzes the reversible condensation of phosphoenolpyruvate and shikimate 3-phosphate to yield 5-enolpyruvyl shikimate 3-phosphate and inorganic phosphate. The enzyme is a target for the nonselective herbicide glyphosate (N-phosphonomethylglycine). In order to determine the role of lysine residues in the mechanism of action of this enzyme as well as in its inhibition by glyphosate, chemical modification studies with pyridoxal 5'-phosphate were undertaken. Incubation of the enzyme with the reagent in the absence of light resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo first-order and saturation kinetics with Kinact of 45 microM and a maximum rate constant of 1.1 min-1. The inactivation rate increased with increase in pH, with a titratable pK of 7.6. Activity of the inactive enzyme was restored by addition of amino thiol compounds. Reaction of enzyme with pyridoxal 5'-phosphate was prevented in the presence of substrates or substrate plus glyphosate, an inhibitor of the enzyme. Upon 90% inactivation, approximately 1 mol of pyridoxal 5'-phosphate was incorporated per mol of enzyme. The azomethine linkage between pyridoxal 5'-phosphate and the enzyme was reduced by NaB3H4. Tryptic digestion followed by reverse phase chromatographic separation resulted in the isolation of a peptide which contained the pyridoxal 5'-phosphate moiety as well as 3H label. By amino acid sequencing of this peptide, the modified residue was identified as Lys-22. The amino acid sequence around Lys-22 is conserved in bacterial, fungal, as well as plant enzymes suggesting that this region may constitute a part of the enzyme's active site.     

Aminolevulinate synthase: lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria. Lysine-313 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5'-phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959-1965). Here we report on the effect of replacement of aminolevulinate synthase lysine-313 by alanine, histidine, and glycine, using site-directed mutagenesis. Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild-type enzyme. Although their absorption spectra indicate that the mutant enzymes bind pyridoxal 5'-phosphate, they bind noncovalently. However, addition of glycine to the mutant enzymes led to the formation of external aldimines. The formation of an external aldimine between the pyridoxal 5'-phosphate cofactor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase-catalyzed reaction. In contrast, lysine-313 is an essential catalytic residue, because the K313-directed mutant enzymes have no measurable activity. In summary, site-directed mutagenesis of the aminolevulinate synthase active-site lysine-313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5'-phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive. This suggests that lysine-313 has a functional role in catalysis.     

Mechanism of the irreversible inactivation of mouse ornithine decarboxylase by alpha-difluoromethylornithine. Characterization of sequences at the inhibitor and coenzyme binding sites.

Mouse ornithine decarboxylase (ODC) was expressed in Escherichia coli and the purified recombinant enzyme used for determination of the binding site for pyridoxal 5'-phosphate and of the residues modified in the inactivation of the enzyme by the enzyme-activated irreversible inhibitor, alpha-difluoromethylornithine (DFMO). The pyridoxal 5'-phosphate binding lysine in mouse ODC was identified as lysine 69 of the mouse sequence by reduction of the purified holoenzyme form with NaB[3H]4 followed by digestion of the carboxymethylated protein with endoproteinase Lys-C, radioactive peptide mapping using reversed-phase high pressure liquid chromatography and gas-phase peptide sequencing. This lysine is contained in the sequence PFYAVKC, which is found in all known ODCs from eukaryotes. The preceding amino acids do not conform to the consensus sequence of SXHK, which contains the pyridoxal 5'-phosphate binding lysine in a number of other decarboxylases including ODCs from E. coli. Using a similar procedure to analyze ODC labeled by reaction with [5-14C]DFMO, it was found that lysine 69 and cysteine 360 formed covalent adducts with the inhibitor. Cysteine 360, which was the major adduct accounting for about 90% of the total labeling, is contained within the sequence -WGPTCDGL(I)D-, which is present in all known eukaryote ODCs. These results provide strong evidence that these two peptides form essential parts of the catalytic site of ODC. Analysis by fast atom bombardment-mass spectrometry of tryptic peptides containing the DFMO-cysteine adduct indicated that the adduct formed in the enzyme was probably the cyclic imine S-(2-(1-pyrroline)methyl)cysteine. This is readily oxidized to S-((2-pyrrole)methyl)cysteine or converted to S-((2-pyrrolidine)methyl)cysteine by NaBH4 reduction. This adduct is consistent with spectral evidence showing that inactivation of the enzyme with DFMO does not entail the formation of a stable adduct between the pyridoxal 5'-phosphate, the enzyme, and the inhibitor.     

Active-site-directed inactivation of wheat-germ aspartate transcarbamoylase by pyridoxal 5''-phosphate.

Treatment of 1 microM wheat-germ aspartate transcarbamoylase with 1 mM-pyridoxal 5'-phosphate caused a rapid loss of activity, concomitant with the formation of a Schiff base. Complete loss of activity occurred within 10 min when the Schiff base was reduced with a 100-fold excess of NaBH4. Concomitantly, one amino group per chain was modified. No further residues were modified in the ensuing 30 min. The kinetics of inactivation were examined under conditions where the Schiff base was reduced before assay. Inactivation was apparently first-order. The pseudo-first-order rate constant, kapp., showed a hyperbolic dependence upon the concentration of pyridoxal 5'-phosphate, suggesting that the enzyme first formed a non-covalent complex with the reagent, modification of a lysine then proceeding within this complex. Inactivation of the enzyme by pyridoxal was 20 times slower than that by pyridoxal 5'-phosphate, indicating that the phosphate group was important in forming the initial complex. Partial protection against pyridoxal phosphate was provided by the leading substrate, carbamoyl phosphate, and nearly complete protection was provided by the bisubstrate analogue, N-phosphonoacetyl-L-aspartate, and the ligand-pair carbamoyl phosphate plus succinate. Steady-state kinetic studies, under conditions that minimized inactivation, showed that pyridoxal 5'-phosphate was also a competitive inhibitor with respect to the leading substrate, carbamoyl phosphate. Pyridoxal 5'-phosphate therefore appears to be an active-site-directed reagent. A sample of the enzyme containing one reduced pyridoxyl group per chain was digested with trypsin, and the labelled peptide was isolated and shown to contain a single pyridoxyl-lysine residue. Partial sequencing around the labelled lysine showed little homology with the sequence surrounding lysine-84, an active-centre residue of the catalytic subunit of aspartate transcarbamoylase from Escherichia coli, whose reaction with pyridoxal 5'-phosphate shows many similarities to the results described in the present paper. Arguably the reactive lysine is conserved between the two enzymes whereas the residues immediately surrounding the lysine are not. The same conclusion has been drawn in a comparison of reactive histidine residues in the two enzymes [Cole & Yon (1986) Biochemistry 25, 7168-7174].     

Sequence of a tryptic peptide from the NADPH binding site of the enoyl reductase domain of fatty acid synthase

Fatty acid synthase from the uropygial gland of goose was inhibited by treatment with pyridoxal 5'-phosphate by selectively modifying a lysine residue at the NADPH binding site of the enoyl reductase domain (A. J. Poulose and P. E. Kolattukudy (1980) Arch. Biochem. Biophys. 201, 313-321). Distribution of radioactivity in tryptic peptides generated from the synthase treated with pyridoxal 5'-phosphate/NaB3H4 in the presence and absence of 2'-monophosphoadenosine-5'-diphosphoribose, which protects the enzyme from inactivation by pyridoxal phosphate, showed that modification of one specific peptide was prevented by the protector. This peptide was purified by a combination of Sephadex G-25 column chromatography, anion-exchange chromatography, and high-performance liquid chromatography. The primary structure of this peptide is Val-Phe-Thr-Thr-Val-Gly-Ser-Ala-Glu-Lys(Pxy)-Arg.     

Aldose reductase from human psoas muscle. Affinity labeling of an active site lysine by pyridoxal 5'-phosphate and pyridoxal 5'-diphospho-5'-adenosine

The reaction of aldose reductase from human psoas muscle with either pyridoxal 5'-phosphate (PLP) or pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) results in a pseudo first-order 2-fold activation of the enzyme with the stoichiometric incorporation of 1 mol of either reagent per mol of enzyme. However, in addition to an increase in Vmax there was also an increase in Km for both substrate, DL-glyceraldehyde, and coenzyme, NADPH. This resulted in an overall decrease in catalytic efficiency (kcat/Km). Spectral analysis indicated that activation by both PLP and PLP-AMP was accompanied by Schiff's base formation and epsilon-pyridoxyllysine was identified in hydrolysates of the reduced enzyme PLP-complex. Digestion of either PLP-modified or PLP-AMP-modified aldose reductase with endoproteinase Lys-C followed by high performance liquid chromatography purification and amino acid sequencing of the pyridoxyllated peptide revealed that PLP and PLP-AMP had modified the same lysine residue. A 32-residue peptide containing the essential lysine was found to be highly homologous with a segment of the sequence of both human liver aldehyde reductase and rat lens aldose reductase. A tetrapeptide (Ile-Pro-Lys-Ser) containing the essential lysine was identical in all three enzymes. These results highlight the close structural similarity between members of the aldehyde reductase family.     

Amino acid sequence around the pyridoxal 5'-phosphate binding site of rat liver L-threonine deaminase

Rat liver L-threonine deaminase is a pyridoxal 5'-phosphate dependent enzyme. In the present study we have studied the amino acid sequence composition of the peptide surrounding the coenzyme binding lysine at the active site of the native enzyme. We have also examined the homology between this peptide and the analogue forms of L-threonine deaminase from different sources.     

Functional asymmetry for the active sites of linked 5-aminolevulinate synthase and 8-amino-7-oxononanoate synthase

5-Aminolevulinate synthase (ALAS) and 8-amino-7-oxononanoate synthase (AONS) are homodimeric members of the α-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Previously, linking two ALAS subunits into a single polypeptide chain dimer yielded an enzyme (ALAS/ALAS) with a significantly greater turnover number than that of wild-type ALAS. To examine the contribution of each active site to the enzymatic activity of ALAS/ALAS, the catalytic lysine, which also covalently binds the PLP cofactor, was substituted with alanine in one of the active sites. Albeit the chemical rate for the pre-steady-state burst of ALA formation was identical in both active sites of ALAS/ALAS, the k(cat) values of the variants differed significantly (4.4±0.2 vs. 21.6±0.7 min(-1)) depending on which of the two active sites harbored the mutation. We propose that the functional asymmetry for the active sites of ALAS/ALAS stems from linking the enzyme subunits and the introduced intermolecular strain alters the protein conformational flexibility and rates of product release. Moreover, active site functional asymmetry extends to chimeric ALAS/AONS proteins, which while having a different oligomeric state, exhibit different rates of product release from the two ALAS and two AONS active sites due to the created intermolecular strain.     

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.     

Biosynthetic alanine racemase of Salmonella typhimurium: purification and characterization of the enzyme encoded by the alr gene

An alanine racemase, encoded by the alr (dal) gene and believed to be the biosynthetic source of D-alanine for cell wall formation, was purified to homogeneity from an overproducing strain of Salmonella typhimurium (dadB), and the enzymological properties of this enzyme were compared with those of the dadB alanine racemase that functions in the catabolism of L-alanine [Wasserman, S. A., Daub, E., Grisafi, P., Botstein, D., & Walsh, C. T. (1984) Biochemistry 23, 5182]. The alr-encoded enzyme has a monomeric structure with a molecular weight of about 40 000. One mole of pyridoxal 5'-phosphate is bound per mole of enzyme, which is essential for catalytic activity of the enzyme. After the internal Schiff base with pyridoxal 5'-phosphate was reduced with NaB3H4, followed by carboxamidomethylation and tryptic digestion of the enzyme, the amino acid sequence of the pyridoxal 5'-phosphate binding peptide was determined. The sequence of 10 amino acid residues around the lysine residue, to which pyridoxal 5'-phosphate is bound, was identical with that of the dadB racemase. No homology was found in the amino-terminal amino acid sequence between the two enzymes. The enzyme was inactivated with D- and L-beta-fluoroalanine, D- and L-beta-chloroalanine, and D-O-acetylserine in a mechanism-based fashion with a common partition ratio of about 150. The enzyme was labeled with an equimolar amount of [14C]-D-beta-chloroalanine. The inactivator-pyridoxal 5'-phosphate adduct was isolated and shown to be the same structure formed in the dadB racemase inactivation [Roise, D., Soda, K., Yagi, T., & Walsh, C. (1984) Biochemistry 23, 5195].     

Essential Active-Site Lysine of Brain Glutamate Dehydrogenase Isoproteins

Abstract: Two soluble forms of bovine brain glutamate dehydrogenase (GDH) isoproteins were inactivated by pyridoxal 5'-phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through Schiff's base formation with amino groups of the enzyme. Sodium borohydride reduction of the pyridoxal 5'-phosphate-inactivated GDH isoproteins produced a stable pyridoxyl enzyme derivative that could not be reactivated by dialysis. The pyridoxyl enzyme was studied through fluorescence spectroscopy. No substrates or coenzymes separately gave complete protection against pyridoxal 5'-phosphate. A combination of 10 m M 2-oxoglutarate with 2 m M NADH, however, gave complete protection against the inactivation. Tryptic peptides of the isoproteins, modified with and without protection, resulted in a selective modification of one lysine. In both GDH isoproteins, the sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other GDH species.     

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.     

Isolation and sequence of the pyridoxal 5'-phosphate active-site peptide from Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was modified with pyridoxal 5'-phosphate and then reduced with sodium borohydride. Both carboxylase and oxygenase activities were lost when one molecule of pyridoxal 5'-phosphate was bound per enzyme dimer. Peptide maps of modified enzyme showed one N6-(phosphopyridoxal)lysine-containing peptide. This peptide was isolated by gel filtration and cation-exchange chromatography and its sequence determined as Ala-Leu-Gly-Arg-Pro-Glu-Val-Asp-(PLP-Lys)-Gly-Thr-Leu-Val-Ile-Lys. Since activation of the enzyme with Mg2+/CO2 enhances pyridoxal 5'-phosphate modification and subsequent inactivation and the substrate ribulose bisphosphate protects against modification, the modified lysyl group is most certainly at the catalytic site and not at the activation site of the enzyme.