Subunit level crosslinking of rabbit muscle pyruvate kinase by o-phthaldialdehyde

o-Phthaldialdehyde caused irreversible inhibition of rabbit muscle pyruvate kinase following preliminary formation of an enzyme-reagent complex. At pH 7.5, 35 degrees C, the dissociation constant for the complex and the maximal pseudo-first-order rate constant for covalent modification were 0.32 +/- 0.08 mM and 2.54 +/- 0.23 min-1, respectively. The inactivation was accompanied by uv-spectral changes pointing to isoindole formation, with a limiting stoichiometry of 1 isoindole linkage per enzyme subunit. Phosphoenolpyruvate, ADP, and ATP effectively protected the enzyme against inactivation, suggesting that the active site is the target of o-phthaldialdehyde action. As native and modified enzymes were indistinguishable with respect to mobility of the major band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was concluded that the crosslinkage was intrasubunit in character, and that the amino acid residues involved must be closely positioned in the polypeptide backbone. Lysine 366, previously shown to be selectively reactive toward 2',3'-dialdehyde ADP (Bezares et al., 1987, Arch, Biochem. Biophys. 253, 133-137), and cysteine 325 or 357 are implicated.     

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.     

Affinity labeling of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase with the 2',3'-dialdehyde derivative of ATP

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is completely inactivated by the 2',3'-dialdehyde derivative of ATP (oATP) in the presence of Mn2+. The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 +/- 17 microM) prior to covalent modification. The maximum inactivation rate constant at pH 7.5 and 30 degrees C is 0.200 +/- 0.045 min-1. ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation. oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site. The partially inactivated enzyme shows an unaltered Km but a decreased V as compared with native phosphoenolpyruvate carboxykinase. Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pKa of 8.1 for the reactive amino acid residue in the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of [8-14C]oATP per mole of enzyme subunit. The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase.     

The presence of reactive SH groups in the enzymatically active dicyano derivative of creatine kinase

It has been shown that the active dicyano derivative of creatine kinase (ATP:creatine N-phosphotransferase) obtained by cyanolysis of the 5,5'-dithiobis(2-nitrobenzoic acid)-modified and inactivated enzyme contains, as does the native enzyme, two reactive SH groups. Modification of these two SH groups leads to complete inactivation of the dicyano enzyme. Reaction with 4-iodoacetamido-1-naphthol introduces fluorescent labels at these reactive SH groups of the native and the dicyano enzymes. Following tryptic digestion, the respective fluorescent-labelled peptides have been separated by HPLC and the amino acid composition analysis of these peptides has shown that they are consistent with the sequence of the peptide segment containing the active-site SH of Cys-282 of creatine kinase for both the native and the dicyano enzymes, showing that the active SH groups are free in the dicyano enzyme. Upon mild denaturation in 3 M urea, it can be shown that two of the SH groups partially buried in the native enzyme have been cyanylated in the dicyano enzyme. The two reactive SH groups are therefore essential for the activity of creatine kinase and the two cyanylated SH groups are internal groups which probably contributes partially to the stabilization of an active conformation of the enzyme molecule.     

ATP binding site of mitochondrial creatine kinase. Affinity labelling of Asp-335 with C1RATP

The ATP binding site of mitochondrial creatine kinase from chicken heart has been studied by modifying the purified enzyme with a 14C-labelled ATP analogue, C1RATP, in which the reactive label was covalently bound to the gamma-phosphate group of ATP. The modified enzyme was digested by pepsin, and a single radioactive nonapeptide was isolated by HPLC. Amino acid analysis and direct sequence determination revealed that the isolated peptide corresponds to amino acids 335-343 within the C-terminal region of Mi-CK, this peptide being highly preserved throughout evolution. Asp-335 is very likely the site of modification by C1RATP. The specificity of the ATP analogue for the active site of creatine kinase was demonstrated by the inhibition of the enzymatic activity of Mi-CK by C1RATP and by the prevention of this inhibition bij ADP.     

Affinity modification of creatine kinase from rabbit skeletal muscle by 2',3'-dialdehyde derivatives of ADP and ATP

Periodate-oxidized ADP and ATP (oADP and oATP) are substrates and affinity reagents for creatine kinase from rabbit skeletal muscle. oADP and oATP modified a lysine epsilon-amino group in the nucleotide-binding site of the enzyme. Complete inactivation is observed upon binding 2 moles oADP per 1 mole of the enzyme dimer. Modification with oADP is described by a liner dependence of the log of enzyme activity on time, testifying to a pseudo-first-order of the reaction. The reaction rate constant (ki = 8.10(3) min-1) and dissociation constant for the reversible enzyme-oADP complex (Kd = 62 microM) were determined. ADP protected the enzyme from inactivation and covalent binding of the analog, whereas oADP covalently bound to the enzyme was phosphorylated by phosphocreatine. The data obtained allow to suggest that the epsilon-amino group of a lysine residue of the active site is located in close proximity to ribose of ATP and ADP forming a complex with the enzyme. This group seems essential for correct orientation of the nucleotide polyphosphate chain in the enzyme active center, but take no immediate part in the transphosphorylation process.     

Beta-cyanoalanine synthase: Its purification and basic physico-chemical properties]

A method has been developed for the purification of beta-cyano-L-alanine synthase from etiolated 10-day-old seedlings of blue lupine. High purity preparations of the enzyme were obtained with specific activity exceeding 4000-fold that of the seedling homogenate. Preparations were homogeneous on electrophoresis in polyacrylamide gel. The yield of total activity after purification was approximately 20%. Glutamic acid is the enzyme's only N-terminal amino acid; the molecular weight of the enzyme (both native and treated with 6 M urea) is 52000. The synthase containes one mole of pyridoxal-P per mole of protein; its isoelectric point is situated at pH 4,8. The enzyme's absorption spectrum has a maximum at 410 nm i.e., in the characteristic range of many pyridoxal-U-containing enzymes. Data on the amino acid composition of the enzyme are presented.     

Evidence for a reactive cysteine at the nucleotide binding site of spinach ribulose-5-phosphate kinase

Ribulose-5-phosphate kinase from spinach was rapidly inactivated by N-bromoacetylethanolamine phosphate in a bimolecular fashion with a k2 of 2.0 M-1 S-1 at 2 degrees C and pH 8.0. Ribulose 5-phosphate had little effect on the rate of inactivation, whereas complete protection was afforded by ADP or ATP. The extent of incorporation as determined with 14C-labeled reagent was about 1 molar equivalent per subunit in the presence of ATP with full retention of enzymatic activity, and about 2 molar equivalents per subunit in the completely inactivated enzyme. Amino acid analyses of enzyme derivatized with 14C-labeled reagent reveal that all of the covalently incorporated reagent was associated with cysteinyl residues. Hence two sulfhydryls are reactive, but the inactivation correlates with alkylation of one cysteinyl residue at or near the enzyme's nucleotide binding site. The kinase was also extremely sensitive to the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethyl-maleimide. The reactive sulfhydryl groups are likely those generated by reduction of a disulfide during activation.     

Affinity modification of creatine kinase and ATP-ADP translocase in heart mitochondria: determination of their stoichiometric ratio

The interaction of mitochondrial creatine kinase and ATP-ADP translocase with 2.3-dialdehyde derivatives of ADP and ATP (oADP and oATP) has been studied. It was shown that these compounds are irreversible and specific inhibitors of creatine kinase (KioADP = 0.6mM, KioATP = 1.12 mM) and ATP-ADP translocase (KioADP = 0.065mM, KioATP = 0.14 mM). The substrates protect both enzymes from inactivation by these compounds. The maximal pseudo-first order rate constants for the 2,3-dialdehyde nucleotide derivative interaction with creatine kinase are 0.2 min-1 for oADP (pH 6.5) and 0.11 min-1 for oATP (pH 7.0). A decrease in the creatine kinase activity correlates with the incorporation of the reagent into the protein. The completely inactivated, isolated and purified enzyme contains 1 mol of oADP per mole of active sites. A procedure for simultaneous determination of the creatine kinase and translocase content in mitochondria and mitoplasts has been developed, which is based on the application of [3H]oADP in combination with specific treatment of mitochondria (or mitoplasts) with carboxyatractyloside 2,4-dinitrofluorobenzene and a mixture of creatine kinase substrates (MgADP + phosphocreatine). It has been found that for heart mitochondria from different animals the content of creatine kinase and translocase is 2.1-2.6 and 2.4-2.9 mol per mol of cytochrome c oxidase, respectively. Thus, the stoiochiometric ratio of creatine kinase and ATP-ADP translocase is close to 1.0 for all mitochondrial preparations under study (i.e. rat, dog, rabbit and chicken).     

Photoaffinity labelling of arginine kinase and creatine kinase with a gamma-P-substituted arylazido analogue of ATP

1. An ATP analogue with a photoactivated azide group attached to the gamma-phosphate via an amide bond, ATP gamma-p-azidoanilide, appeared to have potential use as a photoaffinity label for the nucleotide-binding regions of ATP: guanidine phosphotransferases. Upon photolysis in the presence of lobster muscle arginine kinase and rabbit muscle creatine kinase, the analogue is converted to a potent inhibito of these two kinases. This photo-dependent inhibition is specific as it cannot be induced by azidoaniline, a mixture of azidoaniline and ATP or by ATP gamma-p-aminoanilide. Preirradiated under suitable conditions, the photoanalogue still shows a transitory inhibitory effect which, however, slowly vanishes with time (t0.5 = 3 h). 2. The photoinhibition is significantly decreased by the presence of ATP or ADP but is completely prevented by the addition of a mixture of nucleotide and guanidine substrates. Differential spectroscopy and affinity chromatography on Sepharose-ATP demonstrated the inability of photoinactivated arginine kinase and creatine kinase to recognize their nucleotide substrates. 3. Experiments with [14C]ATP gamma-p-azidoanilide indicated that photolysis is associated with an irreversible and stoichiometric binding of the ATP analogue to the enzymes. Autoradiographs made with the peptide maps corresponding to the tryptic digests of each 14C-labelled photomodified enzyme showed an unexpected highly specific labelling of the proteins. 4. Thiiol titrations of the kinases which have been subjected to various photolysis conditions led to the conclusion that the arylnitrene moiety of the photoanalogue is covalently attached to the single reactive cysteinyl side chain present in the active-site region of the two homologous kinases. This amino acid residue appears, therefore, to be located near the phosphate chain binding subsite occupied by the ATP analogue and probably also by the natural nucleotide substrates.     

Mutagenesis of two acidic active site residues in human muscle creatine kinase: implications for the catalytic mechanism

Creatine kinase (CK) catalyzes the reversible phosphorylation of the guanidine substrate, creatine, by MgATP. Although several X-ray crystal structures of various isoforms of creatine kinase have been published, the detailed catalytic mechanism remains unresolved. A crystal structure of the CK homologue, arginine kinase (AK), complexed with the transition-state analogue (arginine-nitrate-ADP), has revealed two carboxylate amino acid residues (Glu225 and Glu314) within 2.8 A of the proposed transphosphorylation site. These two residues are the putative catalytic groups that may promote nucleophilic attack by the guanidine amino group on the gamma-phosphate of ATP. From primary sequence alignments of arginine kinases and creatine kinases, we have identified two homologous creatine kinase acidic amino acid residues (Glu232 and Asp326), and these were targeted for examination of their potential roles in the CK mechanism. Using site-directed mutagenesis, we have made several substitutions at these two positions. The results indicate that of these two residues the Glu232 is the likely catalytic residue while Asp326 likely performs a role in properly aligning substrates for catalysis.     

Purification and localization of brain-type creatine kinase in sodium chloride transporting epithelia of the spiny dogfish, Squalus acanthias.

The targeting of creatine kinase isoenzymes to specific sites within muscle cells provides a system for the regeneration of ATP in situ from ADP and creatine phosphate. We have recently reported the colocalization of brain-type (B) creatine kinase and the nonsarcomeric mitochondrial creatine kinase isoenzymes in the thick ascending limb of the loop of Henle in the rat kidney, suggesting that creatine kinase may regenerate ATP for sodium transport (Friedman, D.L., and Perryman, M.B. (1991) J. Biol. Chem. 266, 22404-22410). In order to test the hypothesis regarding the association of B creatine kinase with sodium transport, we examined the creatine kinase enzymes in the rectal (salt-secreting) gland of the dogfish shark which contains high levels of the Na+/K(+)-ATPase. The creatine kinase isoform composition was determined by non-denaturing electrophoresis, immunoblotting, protein purification, and amino acid sequence analysis. The results demonstrate both B creatine kinase and mitochondrial creatine kinase proteins are present in the rectal gland, an isoform composition which is the same as in the mammalian kidney. By using a combination of chromatographic techniques, shark B creatine kinase was purified to homogeneity and partial sequence data was obtained from two cyanogen bromide peptide fragments. One of these fragments contains the active site and is identical at all sequenced residues with the corresponding region from the echinoderm sperm flagellar creatine kinase, and is 96% homologous with both chicken and rat B creatine kinase subunits. The other fragment corresponds to a region near the N-terminal of mammalian creatine kinases and is 89% homologous with B creatine kinase from chicken. The localization of these isoforms was examined by immunocytochemistry using subunit specific antisera. Mitochondrial creatine kinase and B creatine kinase immunoreactivity are detected in all tubules, and is restricted to the basal region of the cells, which is the site of the Na+/K(+)-ATPase. The conservation of creatine kinase isoform expression in excretory tissue, and the localization of creatine kinase immunoreactivity in the basal region of the tubule cells, demonstrate that subcellular compartmentation of B creatine kinase may underly the functional coupling of creatine kinase activity with sodium transport.     

A comparative study of essential arginine residues in Gramicidin S synthetase 2 and isoleucyl tRNA synthetase

In gramicidin S synthetase 2 (GS 2) from Bacillus brevis, L-proline, L-valine, L-ornithine, and L-leucine activations to aminoacyl adenylates are progressively inhibited by phenylglyoxal. The inactivation of GS 2 obeys pseudo-first-order kinetics. ATP completely prevents inactivation of GS 2 by phenylglyoxal, whereas amino acids only partially prevent it. In the presence of ATP, four arginine residues per mol of GS 2 are protected from modification by phenylglyoxal as determined by amino acid analysis and the incorporation of [7-14C]phenylgloxal into the enzyme protein, indicating that a single arginine residue is necessary for each amino acid activation. In isoleucyl tRNA synthetase from Escherichia coli, phenylglyoxal inhibits activation of L-isoleucine to isoleucyl adenylate. ATP completely prevents inactivation, although isoleucine only partially prevents it. One arginine residue of isoleucyl tRNA synthetase is protected by ATP from modification by phenylglyoxal, suggesting that a single arginine residue is essential for isoleucine activation. These results support the involvement of arginine residues in ATP binding with GS 2 or isoleucyl tRNA synthetase, and thus indicate that arginine residues of amino acid activating enzymes are essential for the formation of aminoacyl adenylates in both nonribosomal and ribosomal peptide biosynthesis.     

Affinity labelling of 5''-nucleotidases with 5''-p-fluorosulphonylbenzoyladenosine.

5'-Nucleotidases play an important role in the metabolism of nucleosides; for example, the hydrolysis of AMP generates adenosine, which can modulate a variety of cellular functions. We have used the membrane-bound AMPase from chicken gizzard and a secreted form of these enzymes to analyse their modification by the substrate analogue 5'-p-fluorosulphonylbenzoyladenosine (5'-FSBA). 5'-FSBA irreversibly inactivates 5'-nucleotidases by means of covalent modification of the proteins. ATP, a competitive inhibitor of chicken gizzard and snake-venom 5'-nucleotidase, abolished the inactivation by 5'-FSBA, demonstrating that the inactivation was due to the modification of amino acid residues essential for AMPase activity. We have synthesized radioactive 5'-FSBA, which was employed for the radiolabelling of chicken gizzard 5'-nucleotidase. Incorporation of radioactivity was completely abolished in the presence of ATP, which showed that 5'-FSBA acted by the selective modification of amino acid residues at the active site whereas other potential reactive residues of the protein were not attacked. Limited proteolysis of affinity-labelled chicken gizzard 5'-nucleotidase permitted the identification of digestion products containing the catalytic centre. Pseudo-first-order kinetics indicate that modification of a minimum of one amino acid side chain at the active centre is sufficient to result in inactivation of both chicken gizzard and snake-venom 5'-nucleotidases. Incorporation of the radioactive p-sulphonylbenzoyladenosine moiety parallels the inactivation of 5'-nucleotidase by 5'-FSBA and further substantiated the idea that modification of one amino acid residue at the active centre results in loss of the AMPase activity.     

Dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space of rat-heart mitochondria

To investigate whether or not the mitochondrial intermembrane space together with the extramitochondrial space form a homogeneous pool for adenine nucleotides, rat-heart mitochondria were studied in reconstituted systems with pyruvate kinase and ADP-producing enzymes with varied localization. In the hexokinase system, ADP is produced extramitochondrially by added yeast hexokinase, whereas in the creatine kinase system mitochondrial creatine kinase is responsible for ADP regeneration in the intermembrane space. The dependence of mitochondrial respiration on the extramitochondrial [ATP]/[ADP] ratio in both systems was investigated experimentally and by means of computer simulation. Near the resting state, higher [ATP]/[ADP] ratios were found in the creatine kinase system than in the hexokinase system at the same rate of respiration. This and the maintaining of a substantial creatine kinase-stimulated respiration in the presence of pyruvate kinase in excess is explained by a two-compartment model considering diffusion limitations of adenine nucleotides. A diffusion rate constant of (8.7 +/- 4.7) 10(4) microliters X mg-1 X min-1 for ADP and ATP was estimated, resulting in rate-dependent concentration differences up to 13.7 microM AdN between the extramitochondrial space and the AdN-translocator at the maximum rate of oxidative phosphorylation of rat-heart mitochondria. The results support the assumption that ADP diffusion towards the AdN-translocator is limited if its extramitochondrial concentration is low, resulting in a dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space.     

A two-step synthesis of new water-soluble polymers of NAD+ and ADP. The biological properties of these polymers

Alkylation at the N-1 position of the adenine moiety of NAD+, ADP or ATP with 2,3-epoxypropyl acrylate, followed by polymerization with or without acrylamide at pH 8, gave water-soluble polymers of NAD+ and ADP where the alkyl chain was located at the exocyclic adenine C-6 amino group. Cofactor incorporations were good to high: 145-447 mumol NAD+/g polymer and 667 mumol ADP/g polymer. About 30% of the bound NAD+ could be reduced with rabbit muscle lactae dehydrogenase, yeast alcohol dehydrogenase and Bacillus subtilis alanine dehydrogenase; 84% of the bound ADP was phosphorylated with rabbit muscle creatine kinase. High cofactor activities were obtained with polymerized NAD+ with alcohol dehydrogenase as enzyme: the initial rate of NAD+ polymer reduction was 35-81% that of free NAD+. These values remained substantially high with agarose-immobilized alcohol dehydrogenase (15-36%) and should eventually allow their use in continuous enzymatic reactors. Enzymatic phosphorylation of ADP polymer by creatine kinase gave an ATP polymer with high biological activity: 480 mumol ATP/g polymer were transformed with yeast hexokinase.     

A reactive arginine in adenylate kinase.

Adenylate kinase (ATP:AMP phosphotransferase, EV 2.7.4.3) from pig heart is inactivated by the specific arginyl reagent phenylglyoxal. During inactivation two molecules of phenyglyoxal are incorporated into the protein indicating the modification of one of the 11 arginine residues. The modification of other amino acids is ruled out. Chemical modification of this essential residue is prevented by high concentrations of the substrates AMP, ADP and MgATP2-. The protection of the substrates is explained by the formation of a ternary abortive enzyme-substrate complex ESS. The dissociation constants KD = [ES] - [S]/[ESS] are determined from the kinetic data of inactivation and protection.     

Affinity labeling of rabbit muscle pyruvate kinase by 5'-p-fluorosulfonylbenzoyladenosine.

Rabbit muscle pyruvate kinase is irreversibly inactivated upon incubation with the adenine nucleotide analogue, 5'-p-fluorosulfonylbenzoyladenosine. A plot of the time dependence of the logarithm of the enzymatic activity at a given time divided by the initial enzymatic activity(logE/Eo) reveals a biphasic rate of inactivation, which is consistent with a rapid reaction to form partially active enzyme having 54% of the original activity, followed by a slower reaction to yield totally inert enzyme. In addition to the pyruvate kinase activity of the enzyme, modification with 5'-p-fluorosulfonylbenzoyladenosine also disrupts its ability to catalyze the decarboxylation of oxaloacetate and the ATP-dependent enolization of pyruvate. In correspondence with the time dependence of inactivation, the rate of incorporation of 5'-p-[14C]fluorosulfonylbenzoyladenosine is also biphasic. Two moles of reagent per mole of enzyme subunit are bound when the enzyme is completely inactive. The pseudo-first-order rate constant for the rapid rate is linearly dependent on reagent concentration, whereas the constant for the slow rate exhibits saturation kinetics, suggesting that the reagent binds reversibly to the second site prior to modification. The adenosine moiety is essential for the effectiveness of 5'-p-fluorosulfonylbenzoyladenosine, since p-fluorosulfonylbenzoic acid does not inactivate pyruvate kinase at a significant rate. Thus, the reaction of 5'-p-fluorosulfonylbenzoyladenosine with pyruvate kinase exhibits several of the characteristics of affinity labeling of the enzyme. Protection against inactivation by 5'-p-fluorosulfonylbenzoyladenosine is provided by the addition to the incubation mixture of phosphoenolpyruvate. Mg-ADP or Mg2+. In contrast, the addition of pyruvate, Mg-ATP, or ADP and ATP alone has no effect on the rate of inactivation. These observations are consistent with the postulate that the 5'-p-fluorosulfonylbenzoyladenosine specifically labels amino acid residues in the binding region of Mg2+ and the phosphoryl group of phosphoenolpyruvate which is transferred during the catalytic reaction. The rate of inactivation increases with increasing pH, and k1 depends on the unprotonated form of an amino acid residue with pK = 8.5. On the basis of the pH dependence of the reaction of pyruvate kinase with 5'-p-fluorosulfonylbenzoyladenosine and the elimination of cysteine residues as possible sites of reaction, it is postulated that lysyl or tyrosyl residues are the most probably candidates for the critical amino acids.     

Chemical modification of Escherichia coli RNA polymerase by diethyl pyrocarbonate: evidence of histidine requirement for enzyme activity and intrinsic zinc binding

RNA polymerase (RPase) from Escherichia coli contains five subunits (alpha 2 beta beta' sigma) and two intrinsic Zn ions located in the beta and beta' subunits. This enzyme was rapidly inactivated by diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees C. The difference spectrum of the DEP-inactivated and native RPases showed a single peak at 240 nm indicating the formation of N-carbethoxyhistidines. No decrease in absorbance at 278 nm, due to O-carbethoxytyrosine, or modification of amino and sulfhydryl groups was observed. Inactivated RPase with six to nine histidines being modified could be fully reactivated by incubation with 0.5 M hydroxylamine at pH 6.0 and room temperature for 1 h. No structural difference was detected between the native and modified enzymes as evidenced by UV/visible and fluorescence spectra, sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern, or gel filtration properties. Substrate ATP at 0.11 and 1.14 mM concentrations provided, respectively, 25% and 90% protection against DEP inactivation, while template DNA did not. These results suggest that one or more histidine residues is/are in close proximity to the substrate binding site. The pH dependence of the DEP inactivation of RPase suggested the modification of histidine at the active site with a pK value of 6.9. The inactivation of RPase by DEP and the formation of N-carbethoxyhistidine displayed a similar second-order rate constant of approximately 0.9 mM-1 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid requirement of the vanadate effect on the binding of calcium and ATP to the calcium transport ATPase of the sarcoplasmic reticulum

Lipid deprivation of the sarcoplasmic reticulum calcium-transport ATPase neither affects the enzyme's affinity for ATP nor that of calcium. In contrast, vanadate binding is almost completely abolished. Lipid substitution by oleic acid which at a ratio of 0.3 mg/mg protein completely reactivates the calcium-dependent ATP hydrolysis restores vanadate binding. Concomitantly the mutual interactions between vanadate and calcium or ATP and ADP, respectively are restored. The vanadate-induced disappearance of the enzyme's ATP binding sites as well as its high-affinity binding sites for calcium follow the same time course. Conversely, the displacement of vanadate by calcium proceeds in parallel with the recovery of ADP binding. In lipid-restituted preparations as well as in native membranes vanadate induces the disappearance of external high-affinity and simultaneously the appearance of internal low-affinity calcium binding sites.