Human erythrocyte glucose-6-phosphate dehydrogenase. Identification of a reactive lysyl residue labelled with pyridoxal 5'-phosphate

Human erythrocyte glucose-6-phosphate dehydrogenase contains a reactive lysyl residue, which can be labelled with pyridoxal 5'-phosphate. The binding of one mole of pyridoxal 5'-phosphate per mole of enzyme subunit produces substantial inactivation. The substrate glucose-6-phosphate prevents the loss of activity, suggesting that the reaction site is close to the substrate-binding site. A tryptic peptide containing the pyridoxal-5'-phosphate-binding lysyl residue has been isolated and characterised. The reactive lysyl residue has been identified in the glucose-6-phosphate dehydrogenase amino acid sequence. Comparison with glucose-6-phosphate dehydrogenase from other sources shows a high homology with a peptide containing a reactive lysyl residue, isolated from the enzyme from Saccharomyces cerevisiae; glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides also contains a region highly homologous with the sequence around the reactive lysyl residue in the human enzyme. The results of this communication provide the first direct evidence for the association of an essential catalytic function with a specific region of the molecule of human erythrocyte glucose-6-phosphate dehydrogenase.     

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.     

Inactivation of rhodanese by pyridoxal 5'-phosphate.

Pyridoxal 5'-phosphate and other aromatic aldehydes inactivate rhodanese. The inactivation reaches higher extents if the enzyme is in the sulfur-free form. The identification of the reactive residue as an amino group has been made by spectrophotometric determination of the 5'-phosphorylated pyridoxyl derivative of the enzyme. The inactivation increases with pyridoxal 5'-phosphate concentration and can be partially removed by adding thiosulfate or valine. Prolonged dialysis against phosphate buffer also leads to the enzyme reactivation. The absorption spectra of the pyridoxal phosphate - rhodanese complex show a peak at 410 nm related to the Schiff base and a shoulder in the 330 nm region which is probably due to the reaction between pyridoxal 5'-phosphate and both the amino and thiol groups of the enzyme that appear reasonably close to each other. The relationship betweenloss of activity and pyridoxal 5'-phosphate binding to the enzyme shows that complete inactivation is achieved when four lysyl residues are linked to pyridoxal 5'-phosphate.     

An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 +/- 0.025 M-1 min-1. Inactivation by pyridoxal 5'-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7700 +/- 860 M-1 min-1. A second-order rate constant of inactivation for the irreversible reaction catalyzed by the enzyme is 1434 +/- 110 M-1 min-1. Treatment of the enzyme with pyridoxal 5'-phosphate gives incorporation of 1 mol of pyridoxal 5'-phosphate per mole of enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of kobs vs pH suggests this active-site lysine has a pKa of 8.1 and a pH-independent rate constant of inactivation of 47,700 M-1 min-1. The phosphate-containing substrates IDP, ITP, and phosphoenolpyruvate offer almost complete protection against inactivation by pyridoxal 5'-phosphate. Modified, inactive enzyme exhibits little change in Mn2+ binding as shown by EPR. Proton relaxation rate measurements suggest that pyridoxal 5'-phosphate modification alters binding of the phosphate-containing substrates. 31P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme.Mn2+.substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5'-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.     

Affinity labeling of pyridoxal kinase with adenosine polyphosphopyridoxal

Pyridoxal kinase is inactivated by preincubation with the affinity label reagent adenosine tetraphosphate pyridoxal (AP4-PL) at a mixing molar ratio of 5:1 AP4-PL contains structural features of the substrates pyridoxal and ATP. The substrate ATP affords substantial protection against inactivation. The extent of chemical modification by the affinity label was determined by measuring the spectroscopic properties of AP4-pyridoxyl chromophores attached to the enzyme after reduction with NaBH4. The incorporation of 2 mol of the affinity label per enzyme dimer is needed for complete inactivation of the kinase. After chymotryptic digestion of the enzyme modified with AP4-PL and reduced with tritiated NaBH4, only one radioactive peptide absorbing at 325 nm was separated by reverse-phase high performance liquid chromatography. The amino acid sequence of the radioactive peptide, elucidated by Edman degradation, revealed that a specific lysyl residue of monomeric pyridoxal kinase has reacted with the affinity label reagent. It is postulated that the modified lysyl residue is involved in direct interactions with phosphoryl groups of ATP.     

Horse liver alcohol dehydrogenase. A study of the essential lysine residue.

1. The inactivation of horse liver alcohol dehydrogenase by pyridoxal 5'-phosphate in phosphate buffer, pH8, at 10 degrees C was investigated. Activity declines to a minimum value determined by the pyridoxal 5'-phosphate concentration. The maximum inactivation in a single treatment is 75%. This limit appears to be set by the ratio of the first-order rate constants for interconversion of inactive covalently modified enzyme and a readily dissociable non-covalent enzyme-modifier complex. 2. Reactivation was virtually complete on 150-fold dilution: first-order analysis yielded an estimate of the rate constant (0.164min-1), which was then used in the kinetic analysis of the forward inactivation reaction. This provided estimates for the rate constant for conversion of non-covalent complex into inactive enzyme (0.465 min-1) and the dissociation constant of the non-covalent complex (2.8 mM). From the two first-order constants, the minimum attainable activity in a single cycle of treatment may be calculated as 24.5%, very close to the observed value. 3. Successive cycles of modification followed by reduction with NaBH4 each decreased activity by the same fraction, so that three cycles with 3.6 mM-pyridoxal 5'-phosphate decreased specific activity to about 1% of the original value. The absorption spectrum of the enzyme thus treated indicated incorporation of 2-3 mol of pyridoxal 5'-phosphate per mol of subunit, covalently bonded to lysine residues. 4. NAD+ and NADH protected the enzyme completely against inactivation by pyridoxal 5'-phosphate, but ethanol and acetaldehyde were without effect. 5. Pyridoxal 5'-phosphate used as an inhibitor in steady-state experiments, rather than as an inactivator, was non-competitive with respect to both NADH and acetaldehyde. 6. The partially modified enzyme (74% inactive) showed unaltered apparent Km values for NAD+ and ethanol, indicating that modified enzyme is completely inactive, and that the residual activity is due to enzyme that has not been covalently modified. 7. Activation by methylation with formaldehyde was confirmed, but this treatment does not prevent subsequent inactivation with pyridoxal 5'-phosphate. Presumably different lysine residues are involved. 8. It is likely that the essential lysine residue modified by pyridoxal 5'-phosphate is involved either in binding the coenzymes or in the catalytic step. 9. Less detailed studies of yeast alcohol dehydrogenase suggest that this enzyme also possesses an essential lysine residue.     

Modification of hydroxymethylbilane synthase (porphobilinogen deaminase) by pyridoxal 5''-phosphate. Demonstration of an essential lysine residue.

When hydroxymethylbilane synthase (porphobilinogen deaminase) from Euglena gracilis is incubated with pyridoxal 5'-phosphate at pH 7.0 and 0 degree C, it rapidly loses part of its activity. The proportion of activity that remains decreases as the concentration of the modifier increases up to approx. 2mM, above which no further significant inactivation occurs. Dialysis of the partly inactivated enzyme restores its activity, whereas reduction with NaBH4 makes the inactivation permanent. The maximum inactivation achievable from one cycle of the treatment with pyridoxal 5'-phosphate, then with borohydride, is 53 +/- 5%; taking this modified enzyme through second and third cycles causes further loss of activity. The enzyme from Rhodopseudomonas spheroides behaves similarly, but there are quantitative differences. Spectroscopic evidence indicates that the inactivation procedure modifies lysine residues, and labelling studies show that epsilon-N-pyridoxyl-L-lysine is a product when permanently inactivated enzyme is completely hydrolysed. Several lysine residues per molecule of the E. gracilis enzyme are modified by the treatment with pyridoxal 5'-phosphate and borohydride, but only one appears to be essential for enzymic activity, since porphobilinogen protects the enzyme against inactivation and then one fewer lysine residue per molecule of enzyme is affected. It is suggested that, during the biosynthesis of hydroxymethylbilane, the first porphobilinogen unit is covalently bound to the enzyme through the epsilon-amino group of the essential lysine.     

Modification of pig M4 lactate dehydrogenase by pyridoxal 5''-phosphate. Demonstration of an essential lysine residue.

1. Pig M4 lactate dehydrogenase treated in the dark with pyridoxal 5'-phosphate at pH8.5 and 25 degrees C loses activity gradually. The maximum inactivation was 66%, and this did not increase with concentrations of pyridoxal 5'-phosphate above 1 mM. 2. Inactivation may be reversed by dialysis or made permanent by reducing the enzyme with NaBH4. 3. Spectral evidence indicates modification of lysine residues, and 6-N-pyridoxyl-lysine is present in the hydrolsate of inactivated, reduced enzyme. 4. A second cycle of treatment with pyridoxal 5'-phosphate and NaBH4 further decreases activity. After three cycles only 9% of the original activity remains. 5. Apparent Km values for lactate and NAD+ are unaltered in the partially inactivated enzyme. 6. These results suggest that the covalently modified enzyme is inactive; failure to achieve complete inactivation in a single treatment is due to the reversibility of Schiff-base formation and to the consequent presence of active non-covalently bonded enzyme-modifier complex in the equilibrium mixture. 7. Although several lysine residues per subunit are modified, only one appears to be essential for activity: pyruvate and NAD+ together (both 5mM) completely protect against inactivation, and there is a one-to-one relationship between enzyme protection and decreased lysine modification. 8. NAD+ or NADH alone gives only partial protection. Substrates give virtually none. 9. Pig H4 lactate dehydrogenase is also inactivated by pyridoxal 5'-phosphate. 10. The possible role of the essential lysine residue is discussed.     

Modification of pyruvate, phosphate dikinase with pyridoxal 5'-phosphate: evidence for a catalytically critical lysine residue

Pyruvate, phosphate dikinase from Bacteroides symbiosus is strongly inhibited by low concentrations of pyridoxal 5'-phosphate. The inactivation follows pseudo-first-order kinetics over an inhibitor concentration range of 0.1-2 mM. The inactivation is highly specific since pyridoxine and pyridoxamine 5'-phosphate, analogues of pyridoxal 5'-phosphate, which lack an aldehyde group, caused little or no inhibition even at high concentrations. The unreduced dikinase-pyridoxal 5'-phosphate complex displays an absorption maxima near 420 nm, typical for Schiff base formation. Following reduction of the Schiff base with sodium borohydride, N6-pyridoxyllysine was identified in the acid hydrolysate. When the enzyme was incubated in the presence of pyridoxal 5'-phosphate and reducing agent, the ATP/AMP, Pi/PPi, and pyruvate/phosphoenolpyruvate isotopic exchange reactions were inhibited to approximately the same extent, suggesting that the modification of the lysyl moiety causes changes in the enzyme that affect the reactivity of the pivotal histidyl residue. Phosphorylation of the histidyl group appears to prevent the inhibitor from attacking the lysine residue. On the other hand, addition of pyridoxal 5'-phosphate to the pyrophosphorylated enzyme promotes release of the pyrophosphate and yields the free enzyme which is subject to inhibition.     

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.     

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].     

Modification of an essential amino group of phosphoenolpyruvate carboxylase from maize leaves by pyridoxal phosphate and by pyridoxal phosphate-sensitized photooxidation

Phosphoenolpyruvate carboxylase from maize leaves was inactivated by pyridoxal 5'-phosphate in the dark and in the light. A two-step reversible mechanism is proposed for inactivation in the dark, which involves the formation of a noncovalent complex prior to a Schiff base with amino groups of the enzyme. Spectral analysis of pyridoxal 5'-phosphate-modified phosphoenolpyruvate carboxylase showed absorption maxima at 432 and 327 nm, before and after reduction with NaBH4, respectively, suggesting that epsilon-amino groups of lysine residues are the reactive groups in the enzyme. A correlation between spectral data and the maximal inactivation obtained with several concentrations of inhibitor allowed us to establish that the incorporation of 4 mol of pyridoxal 5'-phosphate per mole of holoenzyme accounts for total inactivation. The absence of modifier bound to phosphoenolpyruvate carboxylase when the modification was carried out in the presence of phosphoenolpyruvate and MgCl2 suggests the existence of an essential lysine residue at the catalytic site of the enzyme. Modification of phosphoenolpyruvate carboxylase in the light under an oxygen atmosphere resulted in an irreversible inactivation, which was completely protected by phosphoenolpyruvate and MgCl2. Spectral analysis of the photomodified enzyme showed an absorption peak of 320 nm, suggesting light-mediated addition of a nucleophilic residue (probably an imidazole group) to the pyridoxal 5'-phosphate-lysine azomethine bond.     

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].     

Chemical modification of NADPH-cytochrome P-450 reductase. Presence of a lysine residue in the rat hepatic enzyme as the recognition site of 2'-phosphate moiety of the cofactor

Chemical modification of rat hepatic NADPH-cytochrome P-450 reductase by sodium 2,4,6-trinitrobenzenesulfonate (TNBS) resulted in a time-dependent loss of the reducing activity for cytochrome c. The inactivation exhibited pseudo-first-order kinetics with a reaction order approximately one, and a second-order constant of 4.8 min-1 X M-1. The reducing activities for 2,6-dichloroindophenol and K3Fe(CN)6 were also decreased by TNBS. Almost complete protection of the NADPH-cytochrome P-450 reductase from inactivation by TNBS was achieved by NADP(H), while partial protection was obtained with a high concentration of NADH. NAD, FAD and FMN showed no effect against the inactivation. 3-Acetylpyridine-adenine dinucleotide phosphate, adenosine 2',5'-bisphosphate and 2'AMP protected the enzyme against the chemical modification. Stoichiometric studies showed that the complete inactivation was caused by modification of three lysine residues per molecule of the enzyme. But, under the conditions where the inactivation was almost protected by NADPH, two lysine residues were modified. From those results, we propose that one residue of lysine is located at the binding site of the 2'-phosphate group on the adenosine ribose of NADP(H), and plays an essential role in the catalytic function of the NADPH-cytochrome P-450 reductase.     

Evidence for an essential histidyl residue at the active site of pyridoxamine (pyridoxine)-5'-phosphate oxidase from rabbit liver.

Pyridoxamine (pyridoxine)-5'-phosphate oxidase (EC 1.4.3.5) from rabbit liver is inactivated by diethylpyrocarbonate in an all-or-none fashion with first order kinetics with respect to modifier concentration. The rate of inactivation increases with pH and reflects a group with a pKa of 7.5. Inactivated enzyme is in the holo form with intact FMN. Four histidyls and a cysteinyl residue are modified by excess reagent. The restoration of enzymatic activity by hydroxylamine, the spectrophotometric and colorimetric amino acid analyses, and our previous studies on cysteine modification (Tsuge, H., and McCormick, D.B. (1979) in Flavins and Flavoproteins (Yamano, T., and Yagi, K., eds) Japan Scientific Societies Press, Tokyo, in press) all suggest that inactivation occurs solely by modification of histidine. Analyses by kinetic and statistical methods indicate that three histidines are modified slowly and are not critical for activity, while one histidine is modified nine times more rapidly and accounts for the observed inactivation. Inactivated enzyme shows no significant perturbations in structure, as evidenced by absorption, CD, fluorescence, and gel filtration, but is unable to bind the product, pyridoxal 5'-phosphate. Furthermore, the substrate-competitive inhibitor, pyridoxal 5'-phosphate oxime, protects from inactivation. Hence, diethylpyrocarbonate inactivates this enzyme by modifying a crucial histidyl residue at the substrate/product-binding site.     

Inactivation of brain myo-inositol monophosphate phosphatase by pyridoxal-5'-phosphate

Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.     

Identification of a critical lysine residue at the active site in glyceraldehyde-3-phosphate dehydrogenase of Ehrlich ascites carcinoma cell. Comparison with the rabbit muscle enzyme.

The involvement of the lysine residue present at the active site of Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (Gra3PDH) was investigated by using the lysine specific reagents trinitrobenzenesulfonic acid (TNBS) and pyridoxal phosphate (PP). Both TNBS and PP inactivated EAC cell Gra3PDH with pseudo-first-order kinetics with the rate dependent on modifier concentration. Kinetic analysis, including a Tsou plot, indicated that both TNBS and PP apparently react with one lysine residue per enzyme molecule. Two of the substrates, d-glyceraldehyde-3-phosphate and NAD, and also NADH, the product and competitive inhibitor, almost completely protected the enzyme from inactivation by TNBS. A comparative study of Gra3PDH of EAC cell and rabbit muscle indicates that the nature of active site of the enzyme is significantly different in these two cells. A double inhibition study using 5,5'-dithiobis(2-nitrobenzoic acid) and TNBS and subsequent reactivation of only the rabbit muscle enzyme by dithiothreitol suggested that a cysteine residue of this enzyme possibly reacts with TNBS. These studies on the other hand, confirm that an essential lysine residue is involved in the catalytic activity of the EAC cell enzyme. This difference in the nature of the active site of EAC cell Gra3PDH that may be related to the high glycolysis of malignant cells has been discussed.     

Chemical modification of NADP-isocitrate dehydrogenase from Cephalosporium acremonium evidence of essential histidine and lysine groups at the active site.

NADP-isocitrate dehydrogenase from Cephalosporium acremonium CW-19 has been inactivated by diethyl pyrocarbonate following a first-order process giving a second-order rate constant of 3.0 m-1. s-1 at pH 6.5 and 25 degrees C. The pH-inactivation rate data indicated the participation of a group with a pK value of 6.9. Quantifying the increase in absorbance at 240 nm showed that six histidine residues per subunit were modified during total inactivation, only one of which was essential for catalysis, and substrate protection analysis would seem to indicate its location at the substrate binding site. The enzyme was not inactivated by 5, 5'-dithiobis(2-nitrobenzoate), N-ethylmaleimide or iodoacetate, which would point to the absence of an essential reactive cysteine residue at the active site. Pyridoxal 5'-phosphate reversibly inactivated the enzyme at pH 7.7 and 5 degrees C, with enzyme activity declining to an equilibrium value within 15 min. The remaining activity depended on the modifier concentration up to about 2 mm. The kinetic analysis of inactivation and reactivation rate data is consistent with a reversible two-step inactivation mechanism with formation of a noncovalent enzyme-pyridoxal 5'-phosphate complex prior to Schiff base formation with a probable lysyl residue of the enzyme. The analysis of substrate protection shows the essential residue(s) to be at the active site of the enzyme and probably to be involved in catalysis.     

Reaction of pyridoxal 5'-phosphate with Escherichia coli CoA transferase: evidence for an essential lysine residue.

The acetyl-CoA:acetoacetate CoA-transferase of Escherichia coli was reversibly inactivated by pyridoxal 5′-phosphate. The residual activity of the enzyme was dependent on the concentration of the modifying reagent to a concentration of 5 mm. The maximum level of inactivation was 89%. Kinetic and equilibrium analyses of inactivation were consistent with a two-step process (Chen and Engel, 1975, Biochem. J.149, 619) in which the extent of inactivation was limited by the ratio of first-order rate constants for the reversible formation of an inactive Schiff base of pyridoxal 5′-phosphate and the enzyme from a noncovalent, dissociable complex of the enzyme and modifier. The calculated minimum residual activity was in close agreement with the experimentally determined value. The conclusion that the loss of catalytic activity resulted from modification of a lysine residue at the active site was based on the following data, (a) After incubation with 5 mm pyridoxal 5′-phosphate, 3.95 mol of the reagent was incorporated per mole of free enzyme with 89% loss of activity, while 2.75 mol of pyridoxal 5′-phosphate was incorporated into the enzyme-CoA intermediate with a loss of 10% of catalytic activity; the intermediate was formed in the presence of acetoacetyl-CoA; (b) acid hydrolysis of the modified, reduced enzyme-CoA intermediate yielded a single fluorescent compound that was identified as N⁶-pyridoxyllysine by chromatography in two solvent systems; (c) the enzyme was also protected from inactivation by saturating concentrations of free CoA and ADP but not by adenosine. The results suggested that a lysine residue is involved in the electrostatic binding of the pyrophosphate group of CoA. Carboxylic acid substrate did not protect the enzyme from inactivation.     

Mitochondrial aspartate aminotransferase-independent function of the catalytic binding sites.

The enzyme mitochondrial aspartate aminotransferase from beef liver is a dimer of identical subunits. The enzymatic activity of the resolved enzyme is restored upon addition of the cofactor pyridoxal 5-phosphate. The binding of 1 molecule of cofactor restores 50% of the original enzymatic activity, whereas the binding of a 2nd molecule of cofactor brings about more than 95% recovery of the catalytic activity. Following addition of 1 mol of pyridoxal-5-P per dimer, three forms of the enzyme may exist in solution: apoenzyme-2 pyridoxal 5'-phosphate, apoenzyme-1 pyridoxal 5'-phosphate, and apoenzyme. The enzyme species are separated by affinity chromatography and the following distribution was found: apoenzyme-2 pyridoxal 5'-phosphate/apoenzyme-1 pytidoxal 5'-phosphate/apoenzyme, 2/6/2. Similar distribution was observed after reduction with NaBH4 of the mixture containing apoenzyme and pyridoxal-5-P at a mixing ratio of 1:1. Fluorometric titrations conducted on samples of apoenzyme and apoenzyme-1 pyridoxal 5'-phosphate reveal that the enzyme species display identical affinity towards the inhibitor 4-pyridoxic-5-P (KD equals 1.1 times 10- minus 6 M). It is concluded that the binding of the cofactor to one of the catalytic sites does not affect the affinity of the second site for the inhibitor. These results, obtained by two independent methods, lend strong support to the hypothesis that the two subunits of the enzyme function independently.