Alkylation of estradiol 17 beta-dehydrogenase from human placenta with 3-chloroacetylpyridine--adenine dinucleotide phosphate.

3-Chloroacetylpyridine--adenine dinucleotide phosphate is both active as a hydride acceptor and inactivates estradiol 17 beta-dehydrogenase. This coenzyme analogue behaves like an affinity label. The inactivation kinetics are discussed in relation to those observed with 3-chloroacetylpyridine--adenine dinucleotide. The pH dependence of the rate of inactivation, in combination with determination of the number of reactive cysteine residues, pointed to the alkylation of one cysteine residue/subunit. The stoichiometry was one molecule of dinucleotide per subunit and no cooperativity was detected. When 14C-labeled dinucleotide was used, the 14C label was found mainly in one peptide, accounting for 90% of the incorporated radioactivity, whereas in previous work it had been shown that 3-chloroacetylpyridine--adenine dinucleotide is an affinity reagent which labels three peptides.     

Alkylation of 6-phosphogluconate dehydrogenase from Candida utilis with coenzyme analogues.

The mechanism of the inactivation of 6-phosphogluconate dehydrogenase from Candida utilis with two coenzyme analogues can be differentiated on the basis of kinetic studies and of the properties of the inactivated enzyme. 3-Chloroacetylpyridine--adenine dinucleotide phosphate is clearly an affinity label and 3-choloroacetylpyridine--adenine dinucleotide a second-order reagent. For 3-chloroacetylpyridine--adenine dinucleotide phosphate, there is a loss of one thiol per subunit at complete inactivation whereas for 3-chloroacetylpyridine--adenine dinucleotide 2.7 thiol groups are lost. The fluorescence of the protein is quenched after alkylation by 3-chloroacetylpyridine--adenine dinucleotide phosphate and there is no quenching after the inactivation with 3-chloroacetylpyridine--adenine dinucleotide.     

p-(Bromoacetamido)phenyl uridyl pyrophosphate: an active-site-directed irreversible inhibitor for uridine diphosphate galactose 4-epimerase.

The synthesis of p-(bromoacetamido)phenyl uridyl pyrophosphate (BUP) is described. This compound is an active-site-directed irreversible inhibitor of Escherichia coli UDP-galactose 4-epimerase. The inactivation follows pseudo-first-order kinetics at pH 8.5 in nonnucleophilic buffers, and a saturation effect is seen in the pseudo-first-order rate constant as the concentration of BUP is increased. The half-saturation parameter for BUP in the inactivation is 0.21 +/- 0.02 mM, which compares favorably with the inhibition constant of 0.3 +/- 0.05 mM for BUP acting as a competitive reversible inhibitor of the enzyme. The inactivation rate is slow, however, with a minimum half-time of 12 h at pH 8.5 and 27 degrees C. Both specific alkylation and nonspecific alkylation by BUP occur, but nonspecific alkylation is faster than the inactivation and the rate of inactivation correlates well with the rate of covalent incorporation of one molecule of [14C]BUP at the active site.     

Irreversible inhibition of bovine lung angiotensin I-converting enzyme with p-[N,N-bis(chloroethyl)amino]phenylbutyric acid (chlorambucil) and chlorambucyl L-proline and with evidence that an active site carboxyl group is labeled

Bovine lung angiotensin I-converting enzyme is rapidly and irreversibly inactivated by p-[N,N-bis(chloroethyl)amino]phenylbutyric acid (chlorambucil) and by the chlorambucil derivative of L-proline (chlorambucyl-proline). Chlorambucil is a nitrogen mustard alkylating agent that is used as an antineoplastic drug. At any one concentration, the inactivation is pseudo-first order with time. Inhibition by both substances is active site directed as suggested by the formation of a reversible enzyme-inhibitor complex prior to the alkylation reaction and by the fact that L-Phe-L-Pro, a reversible inhibitor which is competitive with substrate, is also competitive with both irreversible inhibitors in protecting the enzyme against inactivation. The second order rate constant for inactivation increases in the pH range 5-8 and reaches a value of 3.5 X 10(3) M-1 . min-1 for chlorambucil and 4.8 X 10(2) M-1 . min-1 for chlorambucyl-proline. Chlorambucyl [U-14C]L-proline reacts 1:1 with the converting enzyme and the uptake of radioactivity paralleled the loss of enzyme activity with and without protection by Phe-Pro. Once bound, the radioactive chlorambucyl proline was released (as the dihydroxy derivative) by hydroxide ion with a second order rate constant of 2.2 M-1 . min-1 at 25 degrees C. The radioactive label is also removed by hydroxylamine at pH 10. The lability of the irreversibly bound inhibitor in alkali and in hydroxylamine indicates that an ester bond is formed by the alkylation of an aspartic acid or glutamic acid side chain.     

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.     

Reactions of 1-bromo-2-[14C]pinacolone with acetylcholinesterase from Torpedo nobiliana. Effects of 5-trimethylammonio-2-pentanone and diisopropyl fluorophosphate

1-Bromo-2-[14C]pinacolone, (CH3)3C14COCH2Br [( 14C]BrPin), was prepared from [1-14C]acetyl chloride and tert-butylmagnesium chloride with cuprous chloride catalyst, followed by bromination. It was examined as an active-site directed label for acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) (AcChE). AcChE, isolated from Torpedo nobiliana, has k(cat) = (4.00 +/- 0.04).10(3) s-1, Km = 0.055 +/- 0.008 mM in hydrolysis of acetylthiocholine, and k(cat) = (5.6 +/- 0.2).10(3) s-1, Km = 0.051 +/- 0.003 mM in hydrolysis of acetylcholine. BrPin, binding in the trimethyl cavity, acts initially as a reversible competitive inhibitor, Ki = 0.20 +/- 0.09 mM, and, with time, as an irreversible covalently bound inactivator. Introduction of 14C from [14C]BrPin into Torpedo AcChE at pH 7.0 was followed by SDS-PAGE, autoradiography and scintillation counting, in the absence and presence of 5-trimethylammonio-2-pentanone (TAP), a competitive inhibitor (Ki = 0.075 +/- 0.001 mM) isosteric with acetylcholine; 1.8-1.9 14C was incorporated per inactivated enzyme unit at 50% inactivation. TAP retarded inactivation by [14C]BrPin, and prevented introduction of 0.9-1.1 14C per unit of enzyme protected. Prior inactivation of AcChE by BrPin prevents reaction with [3H]diisopropyl fluorophosphate [( 3H]DFP). Prior inactivation by DFP or [3H]DFP does not prevent reaction with [14C]BrPin, and this subsequent reaction with BrPin does not displace the [3H] moiety. [14C]BrPin alkylates a nucleophile in the active site, and this reaction does not alkylate or utilize the serine-hydroxyl.     

Inactivation of yeast alcohol dehydrogenase by a reactive coenzyme analogue: 3-chloroacetyl pyridine adenine dinucleotide

Yeast alcohol dehydrogenase is very rapidly and irreversibly inactivated by 3-chloroacetyl pyridine adenine dinucleotide, a reactive NAD+-analogue (Biellmann et al., 1974, FEBS Lett. 40, 29-32). Kinetic investigations with this compound, and structurally related compounds, show that this inactivation, against which NAD+ provides a complete protection, corresponds to an affinity label. The incorporation of the coenzyme analogue correlates linearly with the enzyme inactivation, the total inactivation corresponding to one mole of inactivator per coenzyme binding site. The pH-dependence of the inactivation rates of the enzyme by this coenzyme analogue and by its reduced form reflects exactly the pH variation of their respective dissociation constants. In spite of a good stability of the label in the non denatured inactivated enzyme, no modified amino-acid residue could be identified. Considering the affinity of this analogue for yeast alcohol dehydrogenase and the strict steric requirements of this enzyme towards its ligands, the nature of the inactivation reaction as well as different possibilities of the loss of the label in the inactivated enzyme are discussed.     

Active-site-directed inhibition of 3-hydroxy-3-methylglutaryl coenzyme A synthase by 3-chloropropionyl coenzyme A

3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1-14C]-3-Chloropropionyl-CoA binds covalently to enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [14C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue.     

Studies of glutamate dehydrogenase. Methionine-169: the preferentially carboxymethylated residue.

In phosphate buffer at pH 7.0, 5,5'-dithio-bis(2-nitrobenzoic acid), N-ethylmaleimide or iodoacetamide do not alter the activity of beef liver glutamate dehydrogenase. Iodoacetate, however, inactivities the enzyme irreversibility by alkylation. Combined addition of the coenzyme NADH and the substrate 2-oxoglutarate or the effector GTP protects against this inactivation. The alkylation reaction is independent of pH between pH 6-9 indicating that amino, imidazole or phenolic groups are probably not involved in this reaction. Titration of the thiol groups, after denaturation of the enzyme, revealed the loss of approximately one group per polypeptide chain. However, this is not due to the exclusive alkylation of a cysteine residue, since alkylation with iodo-[2-14C]acetic acid also labels a methionine residue. 50% of the label is incorporated into methionine-169 and only 7% into cysteine-115, the remaining radioactivity is distributed in minor quantities (4%) in several unidentified residues. A probable cause of the erroneous thiol groups titration is discussed.     

Affinity labeling of a previously undetected essential lysyl residue in class I fructose bisphosphate aldolase.

The affinity label N-bromoacetylethanolamine phosphate (BrAcNHEtOP) has been used previously at pH 6.5 to identify His-359 of rabbit muscle aldolase as an active site residue. We now find that the specificity of the reagent is pH-dependent. At pH 8.5, alkylation with 14C-labeled BrAcNHEtOP abolishes both fructose-1,6-P2 cleavage activity and transaldolase activity. The stoichiometry of incorporation, the kinetics of inactivation, and the protection against inactivation afforded by a competitive inhibitor or dihydroxyacetone phosphate are consistent with the involvement of an active site residue. A comparison of 14C profiles obtained from chromatography on the amino acid analyzer of acid hydrolysates of inactivated and protected samples reveals that inactivation results from the alkylation of lysyl residues. The major peptide in tryptic digests of the inactivated enzyme has been isolated. Based on its amino acid composition and the known sequence of aldolase, Lys-146 is the residue preferentially alkylated by the reagent. Aldolase modified at His-359 is still subject to alkylation of lysine; thus Lys-146 and His-359 are not mutually exclusive sites. However, aldolase modified at Lys-146 is not subject to alkylation of histidine. One explanation of these observations is that modification of Lys-146 abolishes the binding capacity of aldolase for substrates and substrate analogs (BrAcNHEtOP), whereas modification of his-359 does not. Consistent with this explanation is the ability of aldolase modified at His-359 to form a Schiff base with substrate and the inability of aldolase modified at Lys-146 to do so. Therefore, Lys-146 could be one of the cationic groups that functions in electrostatic binding of the substrate's phosphate groups.     

Affinity-labelling of the anti-inflammatory drug and prostaglandin-binding site of 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol with 17 beta- and 21-bromoacetoxysteroids.

The homogeneous 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol binds prostaglandins with low micromolar affinity at its active site and is competitively inhibited by the non-steroidal and steroidal anti-inflammatory drugs [Penning, Mukharji, Barrows & Talalay (1984) Biochem. J. 222, 601-611]. To examine the portion of this binding site that accommodates the glucocorticoid side chain, we have synthesized 17 beta-bromoacetoxy-5 alpha-dihydrotestosterone (BrDHT) and 21-bromoacetoxydesoxycorticosterone (BrDOC) as affinity-labelling agents. Both these agents promote rapid inactivation of the purified enzyme in a time- and concentration-dependent manner. Analyses of the inactivation progress curves gave estimates of Ki for the inactivators and half-life (t1/2) for the enzyme at saturation (tau) as follows: Ki = 33 microM and tau = 18 s for BrDHT, and Ki = 10 microM and tau = 203 s for BrDOC. Under initial-velocity conditions BrDHT and BrDOC act as competitive inhibitors, yielding Ki values identical with those measured in the inactivation experiments. Both indomethacin and prostaglandin E2 can protect the enzyme from inactivation, yielding Ki values for these ligands consistent with those measured independently by competitive-inhibition studies. These data confirm that the bromoacetoxysteroids label the active site, which is coincident with the prostaglandin- and anti-inflammatory-drug-binding site. Neither gel filtration nor extensive dialysis restores activity to the enzyme inactivated with either affinity-labelling agent. Use of radioactive BrDHT or BrDOC, in which either the steroid portion is labelled with 3H or the bromoacetate portion is labelled with 14C, indicates that inactivation is accompanied by a stoichiometric incorporation of 0.7-1.0 molecules of inhibitor per enzyme monomer. The linkage that forms between the dehydrogenase with either [14C]BrDHT or [14C]BrDOC is stable to acid and base treatment. Complete acid hydrolysis of the enzyme inactivated with [14C]BrDHT, followed by amino acid analyses, indicates that 87% of the radioactivity is eluted with carboxymethylcysteine. An almost identical result is obtained with [14C]BrDOC, where at least 75% of the radioactivity is eluted with this amino acid. Thus BrDHT and BrDOC alkylate at least one reactive cysteine residue at the active site that may be of functional importance in binding the glucocorticoid side chain.     

Periodate-oxidized 3-aminopyridine adenine dinucleotide phosphate as a fluorescent affinity label for pigeon liver malic enzyme

Treatment of 3-aminopyridine adenine dinucleotide phosphate with sodium periodate resulted in oxidation of the ribose linked to 3-aminopyridine ring and cleavage of the dinucleotide into 3-aminopyridine and adenosine moieties. These two moieties were separated by thin layer chromatography and were synergistically bound to pigeon liver malic enzyme (EC 1.1.1.40), causing inactivation of the enzyme. The inactivation showed saturation kinetics. The apparent binding constant for the reversible enzyme-reagent binary complex (KI) and the maximum inactivation rate constant at saturating reagent concentration (kmax) were found to be 1.1 +/- 0.02 mM and 0.068 +/- 0.001 min-1, respectively. L-Malate at low concentration enhanced the inactivation rate by lowering the KI value whereas high malate concentration increased the kmax. Mn2+ or NADP+ partially protected the enzyme from the inactivation and gave additive protection when used together. L-Malate eliminated the protective effect of NADP+ or Mn2+. Maximum and synergistic protection was afforded by NADP+, Mn2+ plus L-malate (or tartronate). Oxidized and cleaved 3-aminopyridine adenine dinucleotide phosphate was also found to be a competitive inhibitor versus NADP+ in the oxidative decarboxylation reaction catalyzed by malic enzyme with a Ki value of 4.1 +/- 0.1 microM. 3-Aminopyridine adenine dinucleotide phosphate or its periodate-oxidized cleaved products bound to the enzyme anticooperatively. Oxidized 3-aminopyridine adenine dinucleotide phosphate labeled the nucleotide binding site of the enzyme with a fluorescent probe which may be readily traced or quantified. The completely inactivated enzyme incorporated 2 mol of reagent/mol of enzyme tetramer. The inactivation was partially reversible by dilution and could be made irreversible by treating the modified enzyme with sodium borohydride. This fluorescent compound and its counterpart-oxidized 3-aminopyridine adenine dinucleotide may be a potential affinity label for all other NAD(P)+-dependent dehydrogenases.     

Affinity labelling of the estrogen binding site of glutamate dehydrogenase with iodoacetyldiethylstilbestrol. Selective alkylation of cysteine-89.

Iodoacetyldiethylstilbestrol was used as an affinity label to alkylate the estrogen binding site of bovine liver glutamate dehydrogenase. This reagent induced inactivation and alkylation of the enzyme. The non-alkylating analogues diethylstilbestrol and estradiol protected the enzyme towards alkylation. The apparent constant of alkylation was of the order of magnitude of I50 for the allosteric inhibition by diethylstilbestrol. These two results suggest that alkylation occurred at the estrogen binding site. The stoichiometry of alkylation was between one and two, depending on the experimental conditions. When the stoichiometry was found to be less than or equal to 1, 90% of the label was bound on cystein residues, 70% of which was carried by cysteine-89, a cysteine residue which is known to be inacessible to iodoacetamide in phosphate buffer in the same conditions of temperature and pH.     

Identification of essential lysyl and cysteinyl residues in spinach ribulosebisphosphate carboxylase/oxygenase modified by the affinity label N-bromoacetylethanolamine phosphate

We reported earlier (Schloss, J. V., and Hartman, F. C. (1977) Biochem. Biophys. Res. Commun. 77, 230-236) that N-bromoacetylethanolamine phosphate is an affinity label for spinach ribulosebisphosphate carboxylase/oxygenase. We now show inactivation to be correlated directly with the alkylation either of a single lysyl residue (in the presence of Mg2+) or of 2 different cysteinyl residues (in the absence of Mg2+), consistent with the likelihood that these residues are located in the active site region. This proposition is further supported by the demonstration that the residues are protected from alkylation by substrate, a competitive inhibitor, or the transition state analog 2-carboxyribitol bisphosphate. Tryptic peptides that contain the modified residues have been isolated and sequenced. One of the 2 cysteinyl residues that are subject to alkylation is only 3 residues distant in sequence from the lysyl residue modified by bromoacetylethanolamine phosphate. This lysyl residue is identical with 1 of the 2 lysyl residues alkylated by the previously described affinity label, 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate (Stringer, C. D., and Hartman, F. C. (1978) Biochem. Biophys, Res. Commun. 80, 1043-1048).     

Chemical events in chloropropionyl coenzyme A inactivation of acyl coenzyme A utilizing enzymes

Incubation of 3-chloropropionyl-CoA with 3-hydroxy-3-methylglutaryl-CoA synthase results in exchange of the C2 proton with solvent as inactivation of enzyme proceeds. This enzyme is also inhibited by S-acrylyl-N-acetylcysteamine; the limiting rate constant for inactivation by the acrylyl derivative (0.36 min-1) slightly exceeds the value measured for chloropropionyl-CoA (0.31 min-1). These observations support the intermediacy of acrylyl-CoA in the chloropropionyl-CoA-dependent inactivation of hydroxymethylglutaryl-CoA synthase. Inhibition of fatty acid synthase by chloropropionyl-CoA is primarily due to alkylation of a reactive cysteine, although secondary reaction with the enzyme's pantetheinyl sulfhydryl occurs. Modification of fatty acid synthase by S-acrylyl-N-acetylcysteamine occurs at a limiting rate (1.8 min-1) that is comparable to that estimated for chloropropionyl-CoA-dependent inactivation. However, this enzyme lacks the ability to deprotonate C2 of an acyl group such as the chloropropionyl moiety. Since such a step would be required to generate an acrylyl group from chloropropionyl-S-enzyme, it is likely that a typical affinity labeling process accounts for inactivation of fatty acid synthase by chloropropionyl-CoA. HMG-CoA lyase is also inhibited by S-acrylyl-N-acetylcysteamine. In contrast to the ability of this reagent to serve as a mechanism-based inhibitor of hydroxymethylglutaryl-CoA synthase and an affinity label of fatty acid synthase, it acts as a group-specific reagent in modifying HMG-CoA lyase (kappa 2 = 86.7 M-1 min-1).     

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.     

Inactivation of the Lactobacillus leichmannii ribonucleoside triphosphate reductase by 2'-chloro-2'-deoxyuridine 5'-triphosphate: stoichiometry of inactivation, site of inactivation, and mechanism of the protein chromophore formation

The ribonucleoside triphosphate reductase (RTPR) of Lactobacillus leichmannii is inactivated by the substrate analogue 2'-chloro-2'-deoxyuridine 5'-triphosphate (ClUTP). Inactivation is due to alkylation by 2-methylene-3(2H)-furanone, a decomposition product of the enzymic product 3'-keto-2'-deoxyuridine triphosphate. The former has been unambiguously identified as 2-[(ethylthio)methyl]-3(2H)-furanone, an ethanethiol trapped adduct, which is identical by 1H NMR spectroscopy with material synthesized chemically. Subsequent to rapid inactivation, a slow process occurs that results in formation of a new protein-associated chromophore absorbing maximally near 320 nm. The terminal stages of the inactivation have now been investigated in detail. The alkylation and inactivation stoichiometries were studied as a function of the ratio of ClUTP to enzyme. At high enzyme concentrations (0.1 mM), 1 equiv of [5'-3H]ClUTP resulted in 0.9 equiv of 3H bound to protein and 83% inactivation. The amount of labeling of RTPR increased with increasing ClUTP concentration up to the maximum of approximately 4 labels/RTPR, yet the degree of inactivation did not increase proportionally. This suggests that (1) RTPR may be inactivated by alkylation of a single site and (2) decomposition of 3'-keto-dUTP is not necessarily enzyme catalyzed. The formation of the new protein chromophore was also monitored during inactivation and found to reach its full extent upon the first alkylation. Thus, out of four alkylation sites, only one appears capable of undergoing the subsequent reaction to form the new chromophore. While chromophore formation was prevented by NaBH4 treatment, the chromophore itself is resistant to reduction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active site directed inactivation of rat mammary gland fatty acid synthase by 3-chloropropionyl coenzyme A

3-Chloropropionyl coenzyme A (CoA) irreversibly inhibits rat mammary gland fatty acid synthase. Enzyme inactivation proceeds with first-order kinetics. NADPH (150 microM) as well as acetyl-CoA (500 microM) affords protection against inactivation, suggesting that the inhibitor is active site directed. In contrast, malonyl-CoA (500 microM) offers little protection. With chloro [1-14C]propionyl-CoA, stoichiometries of modification that approach one per enzyme protomer (240 kilodaltons) have been measured. When chloropropionyl-[3'-32P]CoA is used for inactivation, modification stoichiometries are less than 10% of the value observed in the 14C labeling experiments, suggesting that acylation of the enzyme occurs. Radioactivity remains associated with the 14C-labeled protein after performic acid oxidation, indicating that another linkage, in addition to the thio ester adduct, is formed during inactivation. Recovery of [( 14C]carboxyethyl)cysteine from digests of the inactivated enzyme indicates that alkylation of an active site cysteine occurs. The cysteamine sulfhydryl of the acyl carrier peptide is clearly not the site of modification. Loss of overall enzyme activity is tightly linked to decreases in the ketoacyl synthase partial reaction. This observation, coupled with the differential protection measured with acetyl-CoA and malonyl-CoA, suggests that the reagent modifies a residue at the active site involved in condensation. While inactivated enzyme shows good ketoacyl reductase activity when S-(acetoacetyl)-N-acetylcysteamine is used as a substrate, only poor activity for this partial reaction is measured when acetoacetyl-CoA is the substrate. This implies that the function of the acyl carrier peptide (ACP) is impaired during the inactivation process.(ABSTRACT TRUNCATED AT 250 WORDS)     

14C2H2- and 14CO2-labeling studies of the de novo synthesis of polypeptides by Nitrosomonas europaea during recovery from acetylene and light inactivation of ammonia monooxygenase.

Incubation of cells of the nitrifying bacterium Nitrosomonas europaea with 14C2H2 results in the covalent attachment of 14C label to a membrane-bound polypeptide of an approximate Mr of 28,000 (Hyman, M.R., and Wood, P.M. (1985) Biochem. J. 227, 719-725). A labeling procedure using 14C2H2 generated from Ba14CO3 has been used to investigate the correlation between the extent of covalent modification of this polypeptide by 14C from 14C2H2 and the level of ammonia oxidizing activity in whole cells. The time-dependent inactivation of ammonia monooxygenase by 14C2H2 resulted in a progressive and saturable incorporation of 14C into a 27-kDa polypeptide. In contrast, the specific, time-dependent and complete inactivation of ammonia monooxygenase by light resulted in concomitant decrease in the ability of cells to incorporate 14C from 14C2H2 into this polypeptide. The 14C2H2 labeling procedure was also used to investigate the recovery of ammonia monooxygenase activity after complete inactivation of pre-existing ammonia monooxygenase by either C2H2 or light. The recovery of ammonia monooxygenase activity was closely correlated with a recovery of ability of cells to incorporate 14C label from 14C2H2 into the 27-kDa polypeptide. This recovery process was energy (NH4+)-dependent and was inhibited by chloramphenicol and rifampicin, implying that de novo protein synthesis was required. Additional polypeptides labeled with 14C from 14CO2 were also identified during recovery from C2H2 or light inactivation of ammonia monooxygenase.