The interaction of an epoxide with yeast alcohol dehydrogenase: evidence for binding and the modification of two active site cysteines by styrene oxide.

Yeast alcohol dehydrogenase is inactivated and alkylated by styrene oxide in a single exponential kinetic process. The concentration dependence of half-times for inactivation indicates the formation of an enzyme inhibitor complex, KI = 2.5 times 10(-2) M at pH 8.0. Reduced nicotinamide adenine dinucleotide (NADH), at a concentration of 3 times 10(-4) M where Kd congruent to 1 times 10(-5) M, has a small effect on kinetic parameters for inactivation. Although benzyl alcohol and acetamide-NADH increase the KI for styrene oxide in a manner consistent with their dissociation constants, substrate also increases the rate of inactivation at high styrene oxide concentrations. The reciprocal of half-times for inactivation, extrapolated to infinite styrene oxide concentration, increases with pH between 7.6 and 9.0, pK congruent to 8.5. The stoichiometry of alkylation by [3H]styrene oxide is 2.2 mol of reagent incorporated/mol of subunit, and is accompanied by the loss of 1.9 mol of sulfhydryl/mol of subunit; prior alkylation with iodoacetamide reduces the stoichiometry to 0.88:1, and increases the rate of labeling. Tryptic digests of enzyme modified with [14C]iodoacetamide or [3H]styrene oxide produce two major peptides which cochromatograph, indicating that styrene oxide and iodoacetamide modify the same cysteine residues. Previous investigators have reported that iodoacetate, iodoacetamide, and butyl isocyanate alkylate either of two reactive cysteines of yeast alcohol dehydrogenase; both cysteines cannot be modified simultaneously [Belke et al. (1974), Biochemistry 13, 3418]. The inactivation of enzyme by p-chloromercuribenzoate (PCMB) is reported here to be accompanied by the incorporation of 2.3 mol of PCMB/mol of enzyme subunits, in analogy with styrene oxide; the planarity of the alkylating agent appears to be an important factor in determining the stoichiometry of labeling.     

Purification and kinetic and structural properties of spinach leaf NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase

NADP-dependent nonphosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9) from spinach leaves has been purified to apparent electrophoretic homogeneity by ammonium sulfate fractionation, molecular sieving on Sephadex G-200, DEAE-cellulose, and 2',5'-ADP-Sepharose affinity chromatography. The purified enzyme exhibited a specific activity of 15 mumol (mg protein)-1 min-1 and was characterized as a homotetramer with a native molecular weight of 195,000. Preincubation of the purified enzyme with NADP+ resulted in an almost twofold increase in enzymatic activity. The rate of activation was slower than the rate of catalysis, indicating that the enzyme has hysteretic properties. This behavior results in a lag phase during activity measurement of the enzyme preincubated without NADP+. Substrate interaction and product inhibition studies suggest a rapid equilibrium random BiBi mechanism for the reaction. Thiol modifying reagents, iodoacetamide and diamide, completely inactivated the purified enzyme. Inactivation by iodoacetamide exhibited pseudo-first-order kinetics with a rate constant of 0.17 min-1. D-Glyceraldehyde 3-phosphate effectively protected the enzyme against inactivation by thiol reagents, suggesting that modification occurred at or near the substrate-binding site. Complete inactivation of the dehydrogenase was correlated with incorporation of 8 mol [1-14C]iodoacetamide/mol enzyme. Total protection afforded by D-glyceraldehyde 3-phosphate against enzyme inactivation by iodoacetamide was correlated with a protection of 4 mol reactive residues/mol enzyme. On the basis of these results it is suggested that one sulfhydryl group per enzyme subunit is essential for catalysis in spinach leaf nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase. A kinetic and molecular mechanism for the reaction is proposed.     

Inactivation of glutamate dehydrogenase and glutamate synthase from Bacillus megaterium by phenylglyoxal, butane-2,3-dione and pyridoxal 5''-phosphate.

Reaction of phenylglyoxal with glutamate dehydrogenase (EC 1.4.1.4), but not with glutamate synthase (EC 2.6.1.53), from Bacillus megaterium resulted in complete loss of enzyme activity. NADPH alone or together with 2-oxoglutarate provided substantial protection from inactivation by phenylglyoxal. Some 2mol of [14C]Phenylglyoxal was incorporated/mol of subunit of glutamate dehydrogenase. Addition of 1mM-NADPH decreased incorporation by 0.7mol. The Ki for phenylglyoxal was 6.7mM and Ks for competition with NADPH was 0.5mM. Complete inactivation of glutamate dehydrogenase by butane-2,3-dione was estimated by extrapolation to result from the loss of 3 of the 19 arginine residues/subunit. NADPH, but not NADH, provided almost complete protection against inactivation. Butane-2,3-dione had only a slight inactivating effect on glutamate synthase. The data suggest that an essential arginine residue may be involved in the binding of NADPH to glutamate dehydrogenase. The enzymes were inactivated by pyridoxal 5'-phosphate and this inactivation increased 3--4-fold in the borate buffer. NADPH completely prevented inactivation by pyridoxal 5'-phosphate.     

Selectivity of modification when latent and activated forms of the chloroplast F1-ATPase are inactivated by 7-chloro-4-nitrobenzofurazan

The characteristics and specificity of inactivation of the chloroplast F1-ATPase (CF1) with 7-chloro-4-nitrobenzofurazan (Nbf-Cl) have been investigated. Inactivation of the octylglucoside-dependent Mg2+-ATPase activity of latent CF1 by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. Following inactivation of CF1 with [14C]Nbf-Cl, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the majority of the radioactive reagent incorporated is present in the beta subunit. Treatment of the enzyme with [14C]Nbf-Cl following dithiothreitol heat activation, led to similar labeling of the beta subunit and substantial incorporation of 14C into the gamma subunit. On complete inactivation, about 4 mol of Nbf-S-Cys is formed per mole of dithiothreitol-heat-activated CF1. Incorporation of 14C into the gamma subunit is prevented by prior treatment of the latent CF1 or of the dithiothreitol-heat-activated CF1 with iodoacetamide. Following incubation of the dithiothreitol-heat-activated CF1 with iodoacetamide, complete inactivation of the octylglucoside-dependent Mg2+-ATPase activity by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. After stabilization of the [14C]Nbf-O-Tyr derivative by treatment with sodium dithionite, a labeled peptide was purified. Automatic Edman degradation of this peptide revealed the sequence V-X-V-P-A-D-(D). The majority of the radioactivity was cleaved in the second cycle, the position occupied in CF1 by Tyr-beta-328, which is homologous to Tyr-beta-311, the residue reactive with Nbf-Cl in the beef heart mitochondrial F1-ATPase. When CF1, modified at Tyr-beta-328 with Nbf-Cl, is incubated at pH 9.0, the Nbf-O-Tyr adduct is hydrolyzed, leading to concomitant recovery of the ATPase activity. In double labeling experiments, two-dimensional isoelectric focusing in the presence of urea followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that 2-azido-ADP, covalently bound at the tight ADP binding site, and the tyrosine modified by [14C]Nbf-Cl are located in different beta subunits.     

Affinity labeling of the catalytic and AMP allosteric sites of 3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase by 5'-p-fluorosulfonylbenzoyladenosine

The nucleotide analogue 5'-p-fluorosulfonylbenzoyladenosine (FSBA) reacts irreversibly with rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase kinase, causing a rapid loss of the AMP activation capacity and a slower inactivation of the catalytic activity. The rate constant for loss of AMP activation is about 10 times higher (kappa 1 = 0.112 min-1) than the rate constant of inactivation (kappa 2 = 0.0106 min-1). There is a good correspondence between the time-dependent inactivation of reductase kinase and the time-dependent incorporation of 5'-p-sulfonylbenzoyl[14C]adenosine ([14C]SBA). An average of 1.65 mol of reagent/mol of enzyme subunit is bound when reductase kinase is completely inactivated. The time-dependent incorporation is consistent with the postulate that covalent reaction of 1 mol of SBA/mol of subunit causes complete loss of AMP activation, whereas reaction of another mole of SBA/mol of subunit would lead to total inactivation. Protection against inactivation by the reagent is provided by the addition of Mg2+, AMP, Mg-ATP, or Mg-AMP to the incubation mixtures. In contrast, addition of ATP, 2'-AMP, or 3'-AMP has no effect on the rate constants. Mg-ATP protects preferentially the catalytic site against inactivation, whereas Mg-AMP at low concentration protects preferentially the allosteric site. Mg-ADP affords less protection than Mg-AMP to the allosteric site when both nucleotides are present at a concentration of 50 microM with 7.5 mM Mg2+. Experiments done with [14C]FSBA in the presence of some protectants have shown that a close correlation exists between the pattern of protection observed and the binding of [14C]SBA. The postulate is that there exists a catalytic site and an allosteric site in the reductase kinase subunit and that Mg-AMP is the main allosteric activator of the enzyme.     

Transcription by T7 RNA polymerase is not zinc-dependent and is abolished on amidomethylation of cysteine-347

T7 RNA polymerase has been purified to homogeneity from an overproducing clone of Escherichia coli containing pAR1219. Preparations have a zinc content as low as 0.01 mol/mol of enzyme and a high specific activity, 300 000-500 000 units/mg. There are no intrinsic zinc sites. Furthermore, extrinsic Zn2+ does not function as an activator. Supplementation of the assay mix with up to 5 mM ethylenediaminetetraacetic acid has little effect on activity while added Zn2+ is strongly inhibitory at concentrations above 10 microM. This monomeric RNA polymerase is not a zinc metalloenzyme, unlike its multimeric bacterial counterparts. Titration of the urea-denatured protein with 5,5'-dithiobis(2-nitrobenzoic acid) reveals that all 12 Cys residues are present in the free sulfhydryl form, 5 of which are readily accessible to reagent in the native enzyme. More preferential labeling of the sulfhydryls can be achieved with low concentrations of [14C]iodoacetamide, where inactivation of the enzyme proceeds with incorporation of approximately 1.2 mol of [14C]iodoacetamide/mol of polymerase. Amidomethylation primarily occurs at Cys-347, with lesser reaction at Cys-723 and Cys-839. Cys-347 and Cys-723 are in segments of the primary sequence containing numerous basic residues. These same segments have previously been implicated in promoter binding, suggesting that both residues are located within or near the active site region.     

Identification of the 5-iodoacetamidofluorescein reporter site on the Na,K-ATPase

5-Iodoacetamidofluorescein (5-IAF) labels the catalytic (alpha) subunit of dog kidney Na,K-ATPase without inhibiting enzymatic activity and is thus a useful fluorescent reporter of enzyme conformation under conditions of enzyme turnover. In this study conditions for labeling a unique sulfhydryl group are described, and this residue is identified in the cDNA-derived sequence. Reaction with iodoacetate (IAA) prior to fluorescent labeling lowers the stoichiometry of 5-IAF incorporation from 2.1 to 1.2 mol/mol alpha beta protomer, and increases the conformationally dependent fluorescence changes by 40-50%, consistent with the elimination of nonspecific labeling. IAA/IAF-enzyme has the same catalytic activity as the IAF-enzyme. In contrast, treatment with iodoacetamide prior to labeling with 5-IAF abolishes all fluorescence responses, although activity is retained. IAA/IAF-enzyme was digested by extensive trypsinolysis, and the fluorescent peptides released from the membrane were purified by high performance liquid chromatography and sequenced. Several fluorescent peptides were found, containing all or part of the sequence Cys-Ile-Glu-Leu-Cys-Cys-Gly-Ser-Val-Lys, corresponding to residues 452-461 in the sheep alpha subunit. The major site of modification is the second of the vicinal cysteine residues, Cys-457. Phenylarsine oxide, a reagent specific for vicinal sulfhydryl groups, prevents fluorophore incorporation, thereby confirming the identification of the IAF site from the sequence data.     

Reaction of neutral endopeptidase 24.11 (enkephalinase) with arginine reagents

The effect of the arginine-specific reagents phenylglyoxal and butanedione on the activity of neutral endopeptidase 24.11 ("enkephalinase") was determined. Inactivation of the enzyme by butanedione is completely protected by methionine-enkephalin, but only partially protected by methionine-enkephalinamide. In contrast, phenylglyoxal inactivation of the enzyme exhibits saturation kinetics with a Kd of 20 mM. The enzyme is only partially protected against phenylglyoxal inactivation by both methionine-enkephalin and its amide, indicating that phenylglyoxal reacts at two sites. Reaction of the enzyme with phenylglyoxal in the presence of saturating methionine-enkephalin involves the direct reaction of the reagent with the enzyme-substrate complex. Enzyme treated with butanedione or with phenylglyoxal (at site 1) exhibits a 3-5 decrease in substrate binding with little change in kcat. In contrast, reaction with phenylglyoxal in the presence of saturating methionine-enkephalin shows little change in substrate binding but a 4-fold decrease in kcat. Enzyme inactivation involves the incorporation of approximately 1 mol of phenylglyoxal/enzyme subunit in the absence of methionine-enkephalin and approximately 2.5 mol of phenylglyoxal/enzyme subunit in the presence of saturating methionine-enkephalin. These results suggest that an arginine residue on the enzyme is involved in substrate binding.     

The presence of functional arginine residues in phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is completely inactivated by phenylglyoxal and 2,3-butanedione in borate buffer at pH 8.4, with pseudo-first-order kinetics and a second-order rate constant of 144 min-1 X M-1 and 21.6 min-1 X M-1, respectively. Phosphoenolpyruvate, ADP and Mn2+ (alone or in combination) protect the enzyme against inactivation, suggesting that the modification occurs at or near to the substrate-binding site. Almost complete restoration of activity was obtained when a sample of 2,3-butanedione-inactivated enzyme was freed of excess modifier and borate ions, suggesting that only arginyl groups are modified. The changes in the rate of inactivation in the presence of substrates and Mn2+ were used to determine the dissociation constants for enzyme-ligand complexes, and values of 23 +/- 3 microM, 168 +/- 44 microM and 244 +/- 54 microM were found for the dissociation constants for the enzyme-Mn2+, enzyme-ADP and enzyme-phosphoenolpyruvate complexes, respectively. Based on kinetic data, it is shown that 1 mol of reagent must combine per enzyme active unit in order to inactivate the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of 3-4 mol [7-14C]phenylglyoxal per mol of enzyme subunit. Assuming a stoichiometry of 1:1 between phenylglyoxal incorporation and arginine modification, our results suggest that the modification of only two of the three to four reactive arginine residues per phosphoenolpyruvate carboxykinase subunit is responsible for inactivation.     

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

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

Dialdehyde ATP derivative as an affinity modifier of the Na+,K(+)-ATPase active site

Interaction of Na+,K(+)-ATPase from pig kidney in various conformational states with the dialdehyde analogue of ATP, alpha,alpha-(9-adenyl)-alpha'-D-(hydroxymethyl)diglycolaldehyde triphosphate ester (oATP), has been studied. This interaction leads to an enzyme modification which was shown to be of the affinity type according to the following criteria. 1. oATP can be hydrolyzed by Na+,K(+)-ATPase and prevent inhibition of ATPase activity by gamma-[4-(N-2-chloroethyl-N-methylamino)]benzylamide ATP, indicating that it interacts with Na+,K(+)-ATPase in the enzyme active site. 2. oATP irreversibly inhibits ATP-hydrolyzing activity of Na+,K(+)-ATPase; the extent of inactivation is decreased in the presence of 20 mM ATP and depends on the ion composition of the modification medium. The inhibition and ATP protection are maximal in Na+,Mg2(+)-containing buffer. 3. The value of [14C]oATP incorporation into the alpha subunit is proportional to the degree of enzyme inactivation at low (less than 0.1 mM) concentration of oATP and, on extrapolation to complete inhibition, corresponds to incorporation of 1.05 mol reagent/mol alpha subunit. 4. Tryptic hydrolysis of the isolated oATP-modified alpha subunit and subsequent separation of the peptides revealed only one labelled fragment with a molecular mass of about 10 kDa. Localization of the modified fragment in the alpha-subunit polypeptide chain is discussed. A morpholine-like structure was shown to be formed as a result of the modification.     

Pig kidney dopa decarboxylase: inactivation by iodoacetamide and sequence of the carboxyamidomethylcysteine-containing peptide

Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by iodoacetamide following pseudo-first order reaction kinetics. The apparent first order rate constant for inactivation is proportional to the concentration of iodoacetamide and a second order rate constant of 37 M-1 min-1 is obtained at pH 6.8 and 25 degrees C. Cyanogen bromide fragmentation of iodo(1-14C)acetamide - modified inactivated Dopa decarboxylase followed by trypsin digestion yields a single radioactive peptide. Automated Edman degradation reveals a heptapeptide sequence which contains labeled carboxyamidomethylcysteine. This finding and the results of the incorporation of the label from ido (1-14C)acetamide into the enzyme clearly indicate that the modification of 1 mol of SH per mol of enzyme dimer is responsible for the inactivation process. The labeled peptide, which was located by means of limited proteolysis on the fragment corresponding to the COOH-terminal third of the enzyme, has been aligned with a 7 amino acid stretch of Drosophila enzyme. Although this region appears highly conserved in the Dopa decarboxylase enzymes, the cysteinyl residue is not conserved. This observation together with the spectral binding properties of the iodoacetamide inactivated enzyme argue against a functional role for the modifiable cysteine in the mechanism of action of pig kidney enzyme. It is suggested that the loss of pig kidney decarboxylase activity produced by iodoacetamide modification might be attributable to steric hindrance. This could be due to the presence of the bulky acetamidic group on a cysteine residue at, or near, the active center or in a site of strategic importance to the maintenance of the active site topography.     

Affinity labeling of a glutamyl peptide in the coenzyme binding site of NADP+-specific glutamate dehydrogenase of Salmonella typhimurium by 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate

NADP+-specific glutamate dehydrogenase from Salmonella typhimurium, cloned and expressed in Escherichia coli, has been purified to homogeneity. The nucleotide sequence of S. typhimurium gdhA was determined and the amino acid sequence derived. The nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) reacts irreversibly with the enzyme to yield a partially inactive enzyme. After about 60% loss of activity, no further inactivation is observed. The rate of inactivation exhibits a nonlinear dependence on 2-BDB-T epsilon A-2',5'-DP concentration with kmax = 0.160 min-1 and KI = 300 microM. Reaction of 200 microM 2-BDB-T epsilon A-2',5'-DP with glutamate dehydrogenase for 120 min results in the incorporation of 0.94 mol of reagent/mol of enzyme subunit. The coenzymes, NADPH and NADP+, completely protect the enzyme against inactivation by the reagent and decrease the reagent incorporation from 0.94 to 0.5 mol of reagent/mol enzyme subunit, while the substrate alpha-ketoglutarate offers only partial protection. These results indicate that 2-BDB-T epsilon A-2',5'-DP functions as an affinity label of the coenzyme binding site and that specific reaction occurs at only about 0.5 sites/enzyme subunit or 3 sites/hexamer. Glutamate dehydrogenase modified with 200 microM 2-BDB-T epsilon A-2',5'-DP in the absence and presence of coenzyme was reduced with NaB3H4, carboxymethylated, and digested with trypsin. Labeled peptides were purified by high performance liquid chromatography and characterized by gas phase sequencing. Two peptides modified by the reagent were isolated and identified as follows: Phe-Cys(CM)-Gln-Ala-Leu-Met-Thr-Glu-Leu-Tyr-Arg and Leu-Cys(CM)-Glu-Ile-Lys. These two peptides were located within the derived amino acid sequence as residues 146-156 and 282-286. In the presence of NADPH, which completely prevents inactivation, only peptide 146-156 was labeled. This result indicates that modification of the pentapeptide causes loss of activity. Glutamate 284 in this peptide is the probable reaction target and is located within the coenzyme binding site.     

Reaction of pyruvate kinase with the new nucleotide affinity labels 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate

Two new reactive nucleotides have been synthesized and characterized: 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate (8-BDB-TADP and 8-BDB-TATP). ADP or ATP was converted to 8-thio-ADP (-ATP) via 8-bromo-ADP (-ATP), followed by condensation with 1,4-dibromobutanedione. Rabbit muscle pyruvate kinase is inactivated by both reagents in a biphasic manner with an initial rapid loss of 75% activity, followed by a slow total inactivation. The initial fast reaction with both compounds exhibits nonlinear dependence on reagent concentration, indicating formation of a reversible enzyme-reagent complex prior to covalent attachment. The presence of the gamma-phosphoryl group improves the performance of the affinity label: KI values for the fast phase are similar (about 100 microM), whereas kmax for 8-BDB-TATP is about three times greater than that of 8-BDB-TADP (0.286 min-1 vs 0.0835 min-1). After an 80-min incubation with 175 microM of either reagent, about 2 mol/mol of subunit is incorporated with 76% inactivation caused by 8-BDB-TADP and 97% inactivation by 8-BDB-TATP. Loss of activity is prevented by substrates, with the best protection afforded by a combination of ATP, Mn2+, K+, and phosphoenolpyruvate. Reaction of pyruvate kinase with either compound in the presence of protecting ligands leads to incorporation of about 1 mol of reagent/mol of subunit with only about 15% loss of activity. These results suggest that 8-BDB-TADP and 8-BDB-TATP react with two groups on the enzyme, one of which is at or near the active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of an essential lysine in porcine heart mitochondrial malate dehydrogenase.

The reversible inactivation of porcine heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate yields an irreversible modification upon sodium borohydride reduction. A 200-fold molar excess of pyridoxal-5'-P over enzyme results in inactivation to the extent of 54%, and incorporation of 5.7 mol of inactivator per mol of enzyme. The same inactivation carried out in the presence of 80 mM coenzyme, NADH, produces malate dehydrogenase which is approximately 94% active and contains 4.6 mol of pyridoxal-5'-P per mol of enzyme. The incorporation difference between inactivated and protected samples suggests, for total inactivation, the modification of 2 residues per mol of enzyme (i.e. 1 residue per subunit, or 1 per enzymatic active site). This specificity was confirmed by the isolation of a single pyridoxyl-5'-P-labeled "difference peptide" obtained by comparison of the Dowex 1-X2 elution profiles of tryptic digests of protected and inactivated samples, respectively. Amino acid analysis of the peptide demonstrated the presence of N6-pyridoxyl-L-lysine (Lys(Pyx)), establishing the existence of an essential lysing residue in the active center of malate dehydrogenase. The amino acid sequence of the active center hexapeptide has been determined to be: H2NLys(Pyx)Pro-Gly-Met-Thr-Arg-COOH.     

On the subunit stoichiometry of the F1-ATPase and the sites in it that react specifically with p-fluorosulfonylbenzoyl-5'-adenosine.

During the inactivation of the nucleotide-free F1-ATPase at pH 7.0, by p-fluorosulfonyl[14C]benzoyl-5'-adenosine ([14C]FSBA) in the presence of 20% glycerol, about 4.5 g atoms of 14C are incorporated/350,000 g of enzyme. Isolation of the subunits has shown: (a) over 90% of the incorporated label is associated with the alpha and beta subunits; (b) the amount of label incorporated into the alpha subunit is about 0.5 g atoms/mol which is nonspecifically associated with a number of tyrosine and lysine residues; (c) the amount of radioactivity incorporated into the beta subunit is about 0.9 g atoms/mol which correlates with the degree of inactivation of the enzyme and resides on a single tyrosine residue; (d) up to 2.2 mol of alpha subunit have been isolated from each mole of inactivated enzyme; and (e) about 2 mol of beta subunit have been isolated from each mole of inactivated enzyme. These results account for the incorporation of 4.5 g atoms of 14C which are incorporated/mol of ATPase during inactivation if there are three copies each of the alpha and beta subunit present in the enzyme. It has also been shown that 4-chloro-7-nitrobenzofurazan (NBD-Cl) and FSBA react with different tyrosine residues when they inactivate the ATPase. In addition, it has been shown that the ATPase inactivated with FSBA retains the capacity to bind up to 2.2 mol of [14C]ADP/350,000 g of enzyme.     

Some properties of aldehyde dehydrogenase from sheep liver mitochondria.

Aldehyde dehydrogenase from sheep liver mitochondria was purified to homogeneity as judged by electrophoresis on polyacrylamide gels, and by sedimentation-equilibrium experiments in the analytical ultracentrifuge. The enzyme has a molecular weight of 198000 and a subunit size of 48000, indicating that the molecule is a tetramer. Fluorescence and spectrophotometric titrations indicate that each subunit can bind 1 molecule of NADH. Enzymic activity is completely blocked by reaction of 4mol of 5,5'-dithiobis-(2-nitrobenzoate)/mol of enzyme. Excess of disulfiram or iodoacetamide decreases activity to only 50% of the control value, and only two thiol groups per molecule are apparently modified by these reagents.     

Inactivation of NAD-dependent isocitrate dehydrogenase by affinity labeling of the allosteric ADP site by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate

A new reactive ADP analogue has been synthesized: 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate (2-BDB-TADP). Reaction of ADP with m-chloroperoxybenzoic acid gave ADP 1-oxide, which was treated with NaOH, followed by reaction with carbon disulfide to yield 2-thioadenosine 5'-diphosphate. The final product was synthesized by condensation of 2-thioadenosine 5'-diphosphate with 1,4-dibromobutanedione. Reaction of pig heart NAD-specific isocitrate dehydrogenase with this nucleotide analogue (0.4 mM) causes a time-dependent loss of activity to a limiting value of 75% inactivation. The rate constant for inactivation exhibits a nonlinear dependence on the concentration of 2-BDB-TADP, with kmax = 0.021 min-1 and KI = 0.067 mM. Complete protection against inactivation by 0.2 mM 2-BDB-TADP is provided by ADP + Mn2+, but not by Mn2+ alone, isocitrate, alpha-ketoglutarate, or NAD. Incorporation of 2-BDB-TADP is proportional to the extent of inactivation, reaching 1 mol of reagent/mol of enzyme subunit when the enzyme is maximally inactivated. However, when inactivation is totally prevented by incubation with 2-BDB-TADP in the presence of ADP and Mn2+, 0.5 mol of reagent/mol of subunit is still incorporated, suggesting that inactivation may be attributed to 0.5 mol of reagent/mol of average subunit. In the native enzyme, the Km for total isocitrate is 1.8 mM and is decreased 6-fold to 0.3 mM in the presence of 1 mM ADP, whereas in the modified enzyme, with 25% residual activity, the Km for total isocitrate is about the same in the absence (2.0 mM) or presence (1.8 mM) of ADP. These results indicate that 2-BDB-TADP acts as an affinity label of the ADP allosteric site of NAD-dependent isocitrate dehydrogenase.     

2-[(4-Bromo-2,3-dioxobutyl)thio]adenosine 5'-monophosphate, a new nucleotide analogue that acts as an affinity label of pyruvate kinase

A new reactive adenine nucleotide has been synthesized: 2-[(4-bromo-2,3-dioxobutyl)thio]-adenosine 5'-monophosphate (2-BDB-TAMP). Adenosine 5'-monophosphate 1-oxide was synthesized by reaction of AMP with m-chloroperoxybenzoic acid. Treatment with NaOH followed by reaction with carbon disulfide yielded 2-thioadenosine 5'-monophosphate (TAMP). The final product was generated by reaction of TAMP with 1,4-dibromobutanedione. The structure of 2-BDB-TAMP was determined by UV, 1H NMR, and 13C NMR spectroscopy as well as by bromide and phosphorus analysis. Rabbit muscle pyruvate kinase is inactivated by 2-BDB-TAMP at pH 7.0 and 25 degrees C. The inactivation rate exhibits a nonlinear dependence on the reagent concentration with KI = 0.57 mM. Protection against inactivation is provided by ADP and ATP, in the presence of Mn2+, as well as by phosphoenolpyruvate, in the presence of K+; in addition, partial protection is provided by AMP plus Mn2+. Incubation of pyruvate kinase with 0.075 mM 2-BDB-TAMP for 70 min in the absence of protective ligands leads to incorporation of 1.55 mol of reagent/mol of enzyme subunit when the enzyme is 53% inactive. In the presence of ADP and Mn2+, only 0.96 mol of reagent/mol of subunit is incorporated at 70 min, while the enzyme retains 100% activity. Similar results were obtained in the presence of ATP plus Mn2+. Assuming that the groups modified in the absence of ligands include those modified in the presence of the nucleotides, the 53% inactivation can be attributed to the modification of 0.59 (1.55-0.96) group per enzyme subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical modification of bovine heart mitochondrial malate dehydrogenase. Selective modification of cysteine and histidine.

Bovine mitochondrial malate dehydrogenase (EC 1.1.1.37) was inactivated by the specific modifications of a single histidine residue upon reaction with iodoacetamide. NADH protected against this loss of activity and reaction with the histidine residue, suggesting that the histidine is at the NADH binding site. N-Ethylmaleimide also modified the enzyme by reacting with 1 sulfhydryl residue. The reaction rate with N-ethylmaleimide was increased by decreasing the pH from neutrality or by the addition of urea. NADH protected against the modification of the sulfhydryl group under all the conditions tested, again suggesting active site specificity for this inactivation. This enzyme has a subunit weight of 33,000 and is a dimer. The native malate dehydrogenase will bind only 1 mol of NADH and it is thus assumed that there is only a single active site per dimer.