5-Enolpyruvylshikimate-3-phosphate synthase from Escherichia coli--the substrate analogue bromopyruvate inactivates the enzyme by modifying Cys-408 and Lys-411

In order to identify the essential reactive amino acid residues of 5-enolpyruvylshikimate-3-phosphate synthase, the reaction of the enzyme with its substrate analogue bromopyruvate was investigated. Incubation of the enzyme with bromopyruvate resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first-order and saturation kinetics with a Kinact of 28 microM and a maximum rate constant of 0.31 min-1. The inactivation was prevented by preincubation of the enzyme with the substrates shikimate 3-phosphate, 5-enolpyruvylshikimate 3-phosphate or by the combination of shikimate 3-phosphate plus glyphosate (N-phosphonomethylglycine), an inhibitor of the enzyme. Addition of sodium [3H]borohydride to the reaction mixture had no effect on the rate of inactivation but resulted in the incorporation of 3H label to the modified enzyme. Upon 90% inactivation, approximately 1 mol of bromo[14C]pyruvate was incorporated per mole of enzyme modified in the absence or presence of sodium borohydride. When the enzyme was incubated with bromopyruvate in the presence of sodium [3H]borohydride, approximately 1 mol of 3H label was found to be associated per mole of the modified enzyme. Tryptic digestion of these labeled proteins followed by reverse phase chromatographic separation resulted in the isolation of three radioactive peptides. Analyses of these three peptides indicated that bromopyruvate inactivated the enzyme by modifying Cys-408 and Lys-411, which are conserved in all enzyme sequences studied to date.     

The reaction of N-acetylneuraminate lyase with chloropyruvate (Short Communication)

Chloropyruvate, like bromopyruvate, rapidly inactivates N-acetylneuraminate lyase at pH7.2. At 5 degrees C, 0.5mm-chloropyruvate reacted with the enzyme about ten times as fast as bromopyruvate. In contrast, at pH6.0 and 9 degrees C, chloropyruvate reacted with N-acetylcysteine seven times more slowly than bromopyruvate. A brief (2min) incubation of the enzyme with 1.0mm-chloro[(14)C]pyruvate gave an inactive enzyme in which 4.5 cysteine residues were alkylated per molecule of enzyme. This corresponded to the number of [(14)C]pyruvate residues (3.7) bound to the enzyme by borohydride reduction of the [(14)C]-pyruvate complex, and confirmed the previous suggestion that there is one ;essential' cysteine residue per active site. It is suggested that, for this enzyme, chloropyruvate can be selectively used to alkylate the active-site residues, whereas bromopyruvate cannot. The apparent molecular weight of the enzyme prepared by two slightly different methods was approx. 100000 or 250000. The latter value has been used to calculate the number of pyruvate residues bound to the enzyme.     

Pyruvate carboxylase: affinity labelling of the pyruvate binding site.

The active-site-directed reagent, bromopyruvate has been used to covalently label the pyruvate binding site of pyruvate carboxylase (E.C.6.4.1.1.) isolated from sheep liver. Oxalo-acetate proved to be the most effective reaction component in protecting the enzyme against inactivation; pyruvate was less effective although its efficiency was enhanced by the presence of acetyl CoA. The other reaction components, MgATP^2? and HCO₃^? failed to protect the enzyme against inactivation. Using bromo[2¹⁴C]pyruvate, it was shown that at 100% inactivation, 1.5 pyruvyl residues were bound per mole of biotin and when the reaction was carried out in the presence of acetyl CoA, this ratio was reduced to 1.0. Analysis of pronase digests of the enzyme revealed that more than 90% of the radioactivity was present as carboxy-hydroxyethyl cysteine.     

Catabolite inactivation of biodegradative threonine dehydratase of Escherichia coli.

Incubation of Escherichia coli cells with glucose, pyruvate, and certain other metabolites led to rapid inactivation of inducible biodegradative threonine dehydratase. Analysis with several mutant strains showed that pyruvate, and not a metabolite derived from pyruvate, was capable of inactivating enzyme, and that glucose acted indirectly after being converted to pyruvate. Some other alpha-keto acids such as oxaloacetate and alpha-ketobutyrate (but not alpha-ketoglutarate) were also effective. Inactivation of threonine dehydratase by pyruvate was also observed with purified enzyme preparations. The rates of enzyme inactivation increased with increased concentrations of pyruvate and decreased with increased levels of AMP. Increasing protein concentrations lowered the rates of enzyme inactivation. Dithiothreitol had a large effect on the maximum extent of inactivation of the enzyme by pyruvate; high concentrations of AMP and DTT almost completely counteracted the effect of pyruvate. Gel filtration data showed that pyruvate influenced the oligomeric state of the enzyme by altering the association-dissociation equilibrium in favor of dissociation; the Stokes' radius of the pyruvate-inactivated enzyme was 32 A as compared to 42 A for the untreated enzyme. Reassociation of the dissociated form of the enzyme was achieved by removal of excess free pyruvate by dialysis against buffer supplemented with AMP and DTT. Incubation of threonine dehydratase with [14-C]pyruvate revealed apparent covalent attachment of pyruvate to the enzyme. Strong protein denaturants such as guanidine, urea, and sodium dodecyl sulfate failed to release bound radioactive pyruvate; the molar ratio of firmly bound pyruvate was approximately 1 mol/150,000 g of protein. Pretreatment of the enzyme with p-chloromercuribenzoate and 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) did not reduce the binding of [14-C]pyruvate suggesting no active site SH was involved in the pyruvate-enzyme linkage. Titration of active and pyruvate-inactivated enzyme with Nbs2 indicated that the loss in enzyme activity was not due to oxidation of essential sulfhydryl groups on the enzyme. Based on these data we propose that the mechanism of enzyme inactivation by pyruvate involves covalent attachment of pyruvate to the active oligomeric form of the enzyme followed by dissociation of the oligomer to yield inactive enzyme.     

Mechanism of pigeon liver malic enzyme: kinetics, specificity, and half-site stoichiometry of the alkylation of a cysteinyl residue by the substrate-inhibitor bromopyruvate.

Malic enzyme from pigeon liver is alkylated by the substrate analogue bromopyruvate, resulting in the concomitant loss of its oxidative decarboxylase and oxalacetate decarboxylase activities, but not its ability to reduce alpha-keto acids. The inactivation of oxidative decarboxylase activity follows saturation kinetics, indicating the formation of an enzyme-bromopyruvate complex (K congruent to 8 mM) prior to alkylation. The inactivation is inhibited by metal ions and pyridine nucleotide cofactors. Protection of malic enzyme by the substrates L-malate and pyruvate and the inhibitors tartronate and oxalate requires the presence of the above cofactors, which tighten the binding of these carboxylic acids in accord with the ordered kinetic scheme (Hsu, R. Y., Lardy, H. A., and Cleland, W. W. (1967), J. Biol. Chem. 242, 5315-5322). Bromopyruvate is reduced to L-bromolactate by malic enzyme and is an effective inhibitor of L-malate and pyruvate in the overall reaction. The apparent kinetic constants (90 muM-0.8 mM) are one to two orders of magnitude lower than the half-saturation constant (K) of inactivation, indicating a similar tightening of bromopyruvate binding in the E-NADP+ (NADPH)-Mn2+ (Mg2+)-BP complexes. During alkylation, bromopyruvate interacts initially at the carboxylic acid substrate pocket of the active site, as indicated by the protective effect of substrates and the ability of this compound to form kinetically viable complexes with malic enzyme, particularly as a competitive inhibitor of pyruvate carboxylation with a Ki (90 muM) in the same order as its apparent Michaelis constant of 98 muM. Subsequent alkylation of a cysteinyl residue blocks the C-C bond cleavage step. The incorporation of radioactivity from [14C]bromopyruvate gives a half-site stoichiometry of two carboxyketomethyl residues per tetramer, indicating strong negative cooperativity between the four subunits of equal size, or alternatively the presence of structurally dissimilar active sites.     

Active-site residues of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli. Bromopyruvate inactivation and labeling of glutamate 45

Treatment of pure 2-keto-4-hydroxyglutarate aldolase from Escherichia coli, a "lysine-type," Schiff-base mechanism enzyme, with the substrate analog bromopyruvate results in a time- and concentration-dependent loss of enzymatic activity. Whereas the substrates pyruvate and 2-keto-4-hydroxyglutarate provide greater than 90% protection against inactivation by bromopyruvate, no protective effect is seen with glycolaldehyde, an analog of glyoxylate. Inactivation studies with [14C] bromopyruvate show the incorporation of 1.1 mol of 14C-labeled compound/enzyme subunit; isolation of a radioactive peptide and determination of its amino acid sequence indicate that the radioactivity is associated with glutamate 45. Incubation of the enzyme with excess [14C]bromopyruvate followed by denaturation with guanidine.HCl allow for the incorporation of carbon-14 at cysteines 159 and 180 as well. Whereas the presence of pyruvate protects Glu-45 from being esterified, it does not prevent the alkylation of these 2 cysteine residues. The results indicate that Glu-45 of E. coli 2-keto-4-hydroxyglutarate aldolase is essential for catalytic activity, most likely acting as the amphoteric proton donor/acceptor that is required as a participant in the overall mechanism of the reaction catalyzed.     

Bromopyruvate inactivation of glutamate apodecarboxylase. Kinetics and specificity.

A number of halo carboxylic and dicarboxylic acids were substrate-competitive inhibitors of glutamate decarboxylase, with bromosuccinate, 3-bromopropionate, and iodoacetate having the highest affinity for the enzyme. Some of the halo acids also inactivated the apoenzyme. Bromopyruvate at relatively low concentrations inactivated the apoenzyme irreversibly. The rate of the inactivation of the apodecarboxylase was proportional to bromopyruvate at low concentration and approached a constant rate of inactivation at high bromopyruvate concentration. These data are consistent with a two-step inactivation process in which an enzyme-bromopyruvate complex is formed followed by inactivation. The concentration of bromopyruvate giving the half-maximum rate of inactivation was 6.9 mM, and the maximum rate of inactivation was 1.75 min-1 at pH 4.6 and 23 degrees. Much faster rates of inactivation were obtained at pH 5.96 and 6.44. Phosphate, an inhibitor of pyrisoxal-P binding to the apoenzyme, competitively inhibited the inactivation of the apoenzyme by bromopyruvate. In addition, bromopyruvate inhibited the rate of pyridoxal-P binding to the apoenzyme. Kinetics of the incorporation of bromo[2-14C]pyruvate indicated that complete inactivation was obtained when 1.2 mol of radioactive residue were covalently bound per subunit of apoenzyme. Amino acid analyses demonstrated that a cysteinyl residue was alkylated by the bromopyruvate. The bromopyruvate was evidently interacting nincovalently with a cationic group at or near the pyridoxal-P-binding site, and then was alkylating a nearby cysteinyl residue.     

Bromopyruvate as an active-site-directed inhibitor of the pyruvate dehydrogenase multienzyme complex from Escherichia coli

Bromopyruvate behaves as an active-site-directed inhibitor of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase complex of Escherichia coli. It requires the cofactor thiamin pyrophosphate (TPP) and acts initially as an inhibitor competitive with pyruvate (Ki ca. 90 microM) but then proceeds to react irreversibly with the enzyme, probably with the thiol group of a cysteine residue. E1 catalyzes the decomposition of bromopyruvate, the enzyme becoming inactivated once every 40-60 turnovers. Bromopyruvate also inactivates the intact pyruvate dehydrogenase complex in a TPP-dependent process, but the inhibition is more rapid and is mechanistically different. Under these conditions, bromopyruvate is decarboxylated, and the lipoic acid residues in the lipoate acetyltransferase (E2) component become reductively bromoacetylated. Further bromopyruvate then reacts with the new thiol groups thus generated in the lipoic acid residues, inactivating the complex. If reaction with the lipoic acid residues is prevented by prior treatment of the complex with N-ethylmaleimide in the presence of pyruvate, the mode of inhibition reverts to irreversible reaction with the E1 component. In both types of inhibition of E1, reaction of 1 mol of bromopyruvate/mol of E1 chain is required for complete inactivation, and all the evidence is consistent with reaction taking place at or near the pyruvate binding site.     

Alkylation of acetohydroxyacid synthase I from Escherichia coli K-12 by 3-bromopyruvate: evidence for a single active site catalyzing acetolactate and acetohydroxybutyrate synthesis.

Acetohydroxyacid synthase I (AHAS I) purified from Escherichia coli K-12 was irreversibly inactivated by incubation with 3-bromopyruvate. Inactivation was specific, insofar as bromoacetate and iodoacetate were much less effective than bromopyruvate. Inactivation was accompanied by incorporation of radioactivity from 3-bromo[2-14C]pyruvate into acid-insoluble material. More than 95% of the incorporated radioactivity coelectrophoresed with the 60-kilodalton IlvB subunit of the enzyme through a sodium dodecyl sulfate-polyacrylamide gel; less than 5% coelectrophoresed with the 11.2-kilodalton IlvN subunit. The stoichiometry of incorporation at nearly complete inactivation was 1 mol of 14C per mol of IlvB polypeptide. These data indicate that bromopyruvate inactivates AHAS I by alkylating an amino acid at or near a single active site located in the IlvB subunit of the enzyme. We confirmed that this alkylation inactivated both AHAS reactions normally catalyzed by AHAS I. These results provide the first direct evidence that AHAS I catalyzes both acetohydroxybutyrate and acetolactate synthesis from the same active site.     

Bromopyruvate inactivation of 2-keto-3-deoxy-6-phosphogalactonate aldolase of Pseudomonas saccharophila. Kinetics and stereochemistry.

The enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase of Pseudomonas saccharophila is inactivated by the substrate analog beta-bromopyruvate, which satisfies several criteria of being an active site directed reagent. The inactivation exhibits saturation kinetics, and both bromopyruvate and pyruvate (substrate) compete for free enzyme. Upon prolonged incubation, inactivation is virtually complete. The Kinact for bromopyruvate is 12 mM and the minimum inactivation half-time is 16 min with a k of 0.0433 min minus 1. Bromopyruvate is also a substrate for the enzyme in that 3(R,S)-[3-3H2]bromopyruvate is asymmetrically detritiated by the enzyme yielding 3(S)-[3-3H,H]bromopyruvate concomitant with inactivation. At various concentrations of bromopyruvate which affect the inactivation rate, the ratio of nanomoles of bromopyruvate turned over/unit of enzyme inactivated remains constant averaging 12:1, consistent with both inactivation and catalysis occurring at a single protein site, the catalytic site. The above value does not take into account a possible hydrogen isotope effect and is not thus an absolute value. The stereochemistry of bromopyruvate turnover catalyzed by this enzyme is the same as that for 2-keto-3-deoxy-6-phosphogluconate aldolase of P. putida. This fact provides the first evidence that the pyruvate-specific portions of the two active sites may have evolved from a common precursor.     

Effect of phosphate on the kinetics and specificity of glycation of protein

The glycation (nonenzymatic glycosylation) of several proteins was studied in various buffers in order to assess the effects of buffering ions on the kinetics and specificity of glycation of protein. Incubation of RNase with glucose in phosphate buffer resulted in inactivation of the enzyme because of preferential modification of lysine residues in or near the active site. In contrast, in the cationic buffers, 3-(N-morpholino)propane-sulfonic acid and 3-(N-tris(hydroxymethyl)methyl-amino)-2-hydroxypropanesulfonic acid, the kinetics of glycation of RNase were decreased 2- to 3-fold, there was a decrease in glycation of active site versus peripheral lysines, and the enzyme was resistant to inactivation by glucose. The extent of Schiff base formation on RNAse was comparable in the three buffers, suggesting that phosphate, bound in the active site of RNase, catalyzed the Amadori rearrangement at active site lysines, leading to the enhanced rate of inactivation of the enzyme. Phosphate catalysis of glycation was concentration-dependent and could be mimicked by arsenate. Phosphate also stimulated the rate of glycation of other proteins, such as lysozyme, cytochrome c, albumin, and hemoglobin. As with RNase, phosphate affected the specificity of glycation of hemoglobin, resulting in increased glycation of amino-terminal valine versus intrachain lysine residues. 2,3-Diphosphoglycerate exerted similar effects on the glycation of hemoglobin, suggesting that inorganic and organic phosphates may play an important role in determining the kinetics and specificity of glycation of hemoglobin in the red cell. Overall, these studies establish that buffering ions or ligands can exert significant effects on the kinetics and specificity of glycation of proteins.     

Identification of bound pyruvate essential for the activity of phosphatidylserine decarboxylase of Escherichia coli.

Phosphatidylserine decarboxylase, an intrinsic membrane protein of Escherichia coli, catalyzes the decarboxylation of phosphatidylserine, the final step in the biosynthesis of phosphatidylethanolamine, the principal membrane lipid of this organism. The purified enzyme lacks the absorption spectrum characteristic of pyridoxal-containing enzymes, and it has now been found to contain bound pyruvate, the carbonyl function of which is essential for catalytic activity. The decarboxylase is inactivated by treatment with a number of reagents that attack carbonyl groups, including sodium borohydride. Reduction with tritiated borohydride leads to the introduction of stably bound radioactivity, which, after acid hydrolysis, has been identified as tritiated lactate by several chromatographic procedures and by treatment with lactate dehydrogenase. The enzyme resists inactivation by cyanoborohydride in the absence of substrate, but is readily inactivated by this reagent in the presence of phosphatidylserine. Under the conditions of treatment of neutral pH, cyanoborohydride does not react with carbonyl residues at an appreciable rate, but reduces imino groups much more rapidly. This finding, together with demonstrated dependence of the enzyme upon the carbonyl residue of pyruvate for activity, strongly suggests that a Schiff base is formed by addition of the amino group of phosphatidylserine to the pyruvate residue of the enzyme as an essential step in the action of the decarboxylase.     

Studies on the mechanism of action of histone kinase dependent on adenosine 3':5'-monophosphate. Evidence for involvement of histidine and lysine residues in the phosphotransferase reaction.

The reaction of the phosphate residue transfer catalysed by histone kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) was studied. The phosphotransferase reaction was shown to obey the mechanism of ping-pong bi-bi type. After incubation of the catalytic subunit of histone kinase with [gamma-32P]ATP the incorporation of one mole of [32P]phosphage per mole of protein was observed. The tryptic [32P]phosphohistidine-containing peptide was isolated and its N-terminus and amino acid composition were determined. The 2',3'-dialdehyde derivative of ATP (oATP) was used as the affinity label for the catalytic subunit of cyclic-AMP-dependent histone kinase. The inhibitor formed an alidmine bond with epsilon-amino group of the lysine residue of the active site and was irreversibly bound to the enzyme after reduction by sodium borohydride with concurrent irreversible inactivation of the enzyme. After inactivation, about one mole of 14C-labelled inhibitor was incorporated per mole of the enzyme. ATP effectively protected the catalytic subunit of histone kinase against inactivation by oATP. Tryptic digestion of the enzyme-inhibitor complex led to the isolation of the 14C-labelled peptide of the active site of histone kinase. Basing on these results, the role of histidine and lysine residues in the active site of the catalytic subunit of histone kinase was suggested.     

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.     

The interaction of bromopyruvate with the active site of 2-keto-3-deoxygluconate-6-phosphate aldolase ofPseudomonas saccharophila: Kinetics and stereochemistry

The interaction of bromopyruvate with the active site of 2-keto-3-deoxygluconate-6-P aldolase ofPseudomonas saccharophila was investigated. The reagent inactivates the enzyme, exhibiting saturation kinetics and competition with pyruvate. The minimal inactivation half-time was 6 min, equivalent to a first-order rate constant of 0.115 min^?1. The concentration of bromopyruvate giving the half-maximal inactivation rateK _inact was 50 mM. TheK _s value of pyruvate as a competitive inhibitor was 0.85 mM. The enzyme asymmetrically detritiates (3RS)-[3? ³H ₂ ]bromopyruvate, forming, in water, (3S)-[3-³H,H]bromopyruvate. This stereochemistry is also exhibited by 2-keto-6-deoxygalactonate-6-P aldolase isolated from the same organism as well as the 2-keto-3-deoxygluconate-5-P aldolase ofP. putida. Over a range of [3-³H]bromopyruvate concentrations affecting the inactivation rate, the ratio of nanomoles reagent catalytically turned over per unit of enzyme inactivated remained constant at 14:1, providing evidence that both catalysis and alkylation occur at the same protein site.     

Protein-bound choline is released from the pneumococcal autolytic enzyme during adsorption of the enzyme to cell wall particles.

The inactive precursor form of the pneumococcal autolytic enzyme cloned in Escherichia coli was isolated by affinity chromatography on Sepharose-linked choline. The enzyme was recovered in an electrophoretically pure and activated form by elution from the affinity column with radioactive choline solution. When radioactive choline was used for elutions, the enzyme protein isolated contained protein-bound choline, at approximately 1 mol of choline per mol of enzyme protein, indicating the presence of a single choline recognition site. Radioactive choline remained bound to the enzyme protein during dialysis, precipitation by trichloroacetic acid or ammonium sulfate, and during gel filtration, but not during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the choline-labeled autolysin with pneumococcal cell walls at 0 degrees C resulted in the adsorption of the enzyme to the wall particles and a simultaneous release of free choline from the enzyme protein. It is suggested that the choline molecules that became bound to the enzyme protein during the activation of autolysin are expelled from the choline-binding site and replaced by choline residues from the wall teichoic acid as the autolysin molecules adsorb to their insoluble substrate before the onset of enzymatic wall hydrolysis.     

Chemical modification of human placental glutathione transferase by pyridoxal 5'-phosphate.

Incubation of GST pi from human placenta with 8 mM PLP resulted in a rapid loss of activity during the first 10 min, concomitant with a Schiff base formation. This inactivation was probably due to the formation of a reversible adduct between PLP and the enzyme. After sodium borohydride treatment this adduct was reduced and stabilized. Stoichiometry and peptide isolation studies showed that three lysine residues were modified during reaction of GST and PLP. Protection of the enzyme against inactivation was achieved in the presence of 4 mM GSH suggesting that at least one lysyl residue is associated with the substrate binding site. Peptide mapping by digesting the enzyme with trypsin revealed that lysine shielded by GSH is Lys-127. Our results suggest that this residue may play an important role in enzymatic activity.     

Bromopyruvate, a potential affinity label for octopine dehydrogenase

Bromopyruvate, an analogue of pyruvate, one of the substrates of octopine dehydrogenase, was tested as an inhibitor of the enzyme. Provided both the coenzyme and the second substrate, arginine, were present, bromopyruvate rapidly inactivated the enzyme. This inactivation was irreversible, obeyed pseudo-first order kinetics and exhibited a rate saturation effect. Pyruvate protected the enzyme against inactivation by bromopyruvate and these compounds competed for the same site. Bromopyruvate also behaved as a true substrate for the enzyme. This reagent thus exhibits the kinetic characteristics of a good affinity label for octopine dehydrogenase.     

Use of the sodium borohydride reduction technique to identify a gamma-glutamyl phosphate intermediary in the Escherichia coli glutamine synthetase reaction.

Incubation of unadenylylated Escherichia coli glutamine synthetase with ATP, L-[14C]glutamate and metal ion results in the formation of gamma-glutamyl-P which can under appropriate conditions be reduced by sodium borohydride. The acyl-P compound is formed catalytically as judged by the quantity of radioactive alpha-amino-delta-hydroxyvalerate produced compared to the concentration of enzyme subunits. Formation of the glutamyl-P compound occurs in the presence of magnesium or manganous ions, and the relation of this apparent lack of metal ion specificity with regard to the highly specific Mg2+-supported biosynthetic activity of the unadenylylated form is discussed.     

Pyruvate carboxylase: affinity labelling of the magnesium adenosine triphosphate binding site.

The 2' , 3'-dialdehyde derivative of ATP (oATP) was prepared by periodate oxidation and on the following criteria was considered to be an effective affinity label. The magnesium complex of this derivative (Mg-oATP2) was shown to ba linear competitive inhibitor with respect to MgATP2-in both the acetyl-CoA-dependent and -independent activities of the enzyme but was a non-competitive inhibitor with respect to bicarbonate, and an uncompetitive inhibitor with respect to pyruvate. Mg-oATP was covalently bound to pyruvate carboxylase by reduction using sodium borohydride with concurrent irreversible inactivation of the enzyme...