Essential residues in angiotensin converting enzyme: modification with 1-fluoro-2,4-dinitrobenzene

The peptidase and esterase activities of rabbit pulmonary angiotensin converting enzyme (ACE) are rapidly abolished on reaction with 1-fluoro-2,4-dinitrobenzene (Dnp-F). Inactivation follows first-order kinetics with respect to the reagent and is accompanied by stoichiometric incorporation of 3,5-[3H]Dnp, indicating that the effect is due to a specific modification of the enzyme. Thin-layer chromatography of an acid hydrolysate of the modified enzyme indicates that most of the radioactive label is present as O-Dnp-tyrosine (65 to greater than 95%) and the rest as N epsilon-Dnp-lysine. The pH dependence of the reaction is consistent with modification of either tyrosine or lysine. The presence of a competitive inhibitor effectively protects the enzyme against inactivation by Dnp-F. Acetylation of ACE with N-acetylimidazole also protects the enzyme against modification with Dnp-F. The results indicate the presence of catalytically essential tyrosine and lysine residues at the active site of ACE.     

Reaction of 1-fluoro-2,4-dinitrobenzene and 2,4,6-trinitrobenzenesulphonic acid with membranes of sarcoplasmic reticulum.

Membranes of sarcoplasmic reticulum were labelled with 1-fluoro-2,4-dinitro[3H]benzene at pH 6.5 and with 2,4,6-trinitrobenzenesulphonate at pH 9.2. Conditions were chosen to restrict reaction to amino groups, and the effect of blockings of these groups by methyl acetimidate was determined. All proteins were labelled to some extent by both reagents, but, whereas the trinitrophenylation of both lipid and protein amino groups was almost completely blocked by methyl acetimidate, the dinitrophenylation of the ATPase at pH 6.5 was much less affected. The seven amino groups on the ATPase that were labelled under these conditions did not react with methyl acetimidate. This reagent can therefore be used to enhance the specificity of fluorodinitrobenzene for amino groups in a hydrophobic environment. The amino groups on the minor proteins and on the phospholipids that reacted with fluorodinitrobenzene at pH 6.5 were probably in an aqueous environment, since the reaction was blocked by methyl acetimidate.     

L-lactate-2-monooxygenase. Sequence of peptides containing residues modified by 1-fluoro-2,4-dinitrobenzene

L-Lactate-2-monooxygenase (EC 1.13.12.4) inactivated by 1-fluoro-2,4-dinitrobenzene essentially as described previously (Choong, Y. S., Shepherd, M. G., and Sullivan, P. A. (1978) Biochem. J. 173, 255-262) incorporated 2.8 mol of the dinitrophenyl (DNP) moiety per mole of flavin. The inhibitors 2-methyl lactate or sulfite decreased the incorporation to 0.9 mol of DNP per mole of flavin. Peptide mapping by high performance liquid chromatography of radioactively labeled protein digested with trypsin showed three peaks of radioactivity. DNP-amino acid analysis and peptide sequencing showed that 2 distinct cysteine residues and a histidine residue had been modified. Both cysteine peptides were protected from modification by either of the inhibitors, whereas the histidine was only partially protected. The sum of the 2 cysteine peptides accounted for nearly 1 mole of label per mole of monomer. Since both of the cysteines are protected by inhibitors, they both must be in or near the substrate-binding site of the enzyme and appear to be modified in a mutually exclusive fashion. The histidine, on the other hand, does not lie directly in the substrate-binding site. It is possible that this histidine is the positively charged residue that is postulated to be near the N-1 position of the flavin.     

Chemical modification of Lactobacillus casei thymidylate synthetase with 1-fluoro-2,4-dinitrobenzene and 1,5-difluoro-2,4-dinitrobenzene

Previous studies of the pH dependence of sulfhydryl group modification in thymidylate synthetase (W. A. Munroe, C. A. Lewis and R. B. Dunlap, 1978, Biochem. Biophys. Res. Commun.80, 355–360) suggested that a neighboring general base residue enhanced the nucleophilicity of the catalytic cysteinyl side chain. In an effort to identify the latter residue by active site crosslinking, chemical modification of the enzyme by 1,5-difluoro-2,4-dinitrobenzene was investigated and compared with results of modification by 1-fluoro-2,4-dinitrobenzene. Incubation of enzyme with 1-fluoro-2,4-dinitrobenzene led to rapid inactivation and loss of ability to form ternary complexes. Paper chromatography of the acid hydrolysate of enzyme modified with 1-fluoro-2,4-dinitrobenzene yielded two yellow spots, identified as dinitrophenylenecysteine and dinitrophenylenelysine. Specific active site labeling was indicated by substrate protection with dUMP, by the release of 1.65 of fluoride ion per enzyme dimer during inactivation, and by the fact that 70% of the activity was recovered after incubation of the inactivated enzyme with 2-mercaptoethanol, The results of a similar series of studies with 1,5-difluoro-2,4-dinitrobenzene indicated quite specific active site modification. The equivalents of fluoride ion released during modification, 3.5 per enzyme dimer, and the fact that thiolysis of the totally inactivated enzyme led to a recovery of only 18% of the original activity provided evidence for active site crosslinking with the catalytic cysteine as one of the modification sites. Characterization of the modified enzyme, its yellow acid hydrolysate fragments, and a variety of dinitrophenylene crosslinked models suggested that 1,5-difluoro-2,4-dinitrobenzene had modified the enzyme by crosslinking cysteine and serine residues.     

Mutagenicity of 1-fluoro-2,4-dinitrobenzene is affected by bacterial glutathione

Recently we have shown that Salmonella typhimurium tester strains have high levels of the tripeptide glutathione (GSH) and activity of GSH S-transferases (Summer et al., 1979). In continuation of the GSH-dependent suppression of mutagenicity of 1-chloro-2,4-dinitrobenzene in presence of S9 fraction (Summer et al., 1979), this paper is focused on the GSH-dependent detoxifying capacity of the bacterial tester strains. 1-Fluoro-2,4-dinitrobenzene (FDNB), an electrophilic agent, which is used to identify terminal amino acids in proteins (Sanger reagent), readily reacts with GSH leading to a dose-dependent depletion of bacterial GSH. Additionally, FDNB is a strong mutagen for Salmonella typhimurium TA100, TA1538 and TA98 without metabolic activation.Presumably owing to conjugation with bacterial GSH, FDNB in concentrations which were lower or equal to those of bacterial GSH were found to be not mutagenic. Accordingly, increasing amounts of bacteria in the test system require increasing amounts of FDNB for expression of mutagenicity.     

Reactivity of sarcoplasmic reticulum from rabbit cardiac muscle with 1-fluoro- and 1,5-difluoro- 2,4-dinitrobenzene

Sarcoplasmic reticulum preparations from rabbit cardiac and fast skeletal muscle react differentially with low concentrations of 1-fluoro- and 1,5-difluoro-2,4-dinitrobenzene. Dinitrophenylation of cardiac sarcoplasmic reticulum by 1-fluoro-2,4-dinitrobenzene is not affected by Ca2+ and is limited to the lipoprotein-lipid region. This contrasts sharply with the predominant Ca2+-dependent dinitrophenylation of the ATPase protein of rabbit skeletal sarcoplasmic reticulum by this reagent. Formation of non-serial high mol. wt. oligomers by 1,5-difluoro-2,4-dinitrobenzene is significantly greater in cardiac than in skeletal vesicles. Substrate MgATP2- does not protect rabbit cardiac sarcoplasmic reticulum ATPase activity or Ca2+ uptake from dinitrophenylation when monofunctional and bifunctional reagents are used. Chemical differences in the overall structure of the two kinds of membrane preparations can be ascertained from a comparison of the effects of Ca2+ and MgATP2- on the reactivity of these reagents.     

Determination of chemical properties of individual histidine and tyrosine residues of concanavalin A by competitive labeling with 1-fluoro-2,4-dinitrobenzene

A selective peptide-mapping procedure was devised to purify peptides containing histidine or tyrosine residues from proteolytic digests of concanavalin A (Con A). The protein was modified with maleic anhydride followed by 1-fluoro-2,4-dinitrobenzene (Dnp-F) and then digested with thermolysin. The resulting labeled peptides were separated by high-performance liquid chromatography, and the Dnp-histidine and Dnp-tyrosine peptides were identified by their spectral characteristics. From their amino acid compositions, the labeled peptides could all be assigned within the known sequence. Peptides representing five of the six histidines and all seven tyrosines were obtained. With the same peptide-mapping procedure, the chemical properties (pK and reactivity) of these residues were determined. Samples of concanavalin A at various pH values were labeled with trace amounts of [3H]Dnp-F, in the presence of Gln-Gly as an internal standard. To each sample was added an aliquot of a mixture of [14C]Dnp-Gln-Gly and [14C]Dnp-maleyl-Con A. Portions of each sample were removed, [14C]Dnp-Ala-Ala and epsilon-[14C]Dnp-lysine were added, and the mixtures were hydrolyzed. The various Dnp amino acid derivatives were purified by HPLC. The remainder of each [3H]Dnp sample was maleylated, dinitrophenylated, and digested with thermolysin and separated by HPLC as above. From the 3H/14C ratios of the Dnp amino acid derivatives and the Dnp peptides relative to the ratio of the internal standard, pK and reactivity data were obtained for (a) the average behavior of the lysine, histidine, and tyrosine residues and (b) the individual behavior of the N-terminal alanine residue and the five histidine and seven tyrosine residues in the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spin-labelling of phosphorylase b using a paramagnetic 1-fluoro-2,4-dinitrobenzene derivative

Phosphorylase b (1,4-alpha-D-glucan:1,6-alpha-D-glucan 6-alpha-glucosyltransferase, EC 2.4.1.1) can be specifically spin-labelled at a site essential for the catalytic action of the enzyme. A paramagnetic analogue of 1-fluoro-2,4-dinitrobenzene was synthesized and used as a dinitrophenylating agent. Reaction of phosphorylase b with the paramagnetic probe combined with the thiolysis method, leads to spin-labelling of a single -NH2 group (0.75 groups per subunit) with concomitant loss of 50% of the catalytic activity. Dinitrophenylation does not change the sedimentation profile of the enzyme. The ESR spectrum of modified phosphorylase b indicates that the attached label has rather limited segmental mobility and its environment is slightly hydrophobic. Small but subtle conformational changes induced by ligands in this critical site of the macromolecule can be directly detected by the spin-label. Also, sulfhydryl group modification of the spin-labelled enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) has a pronounced effect on the resonance spectrum.     

A re-examination of the reaction between ox liver glutamate dehydrogenase and 1-fluoro-2,4-dinitrobenzene.

Reaction of ox liver glutamate dehydrogenase with 1-fluoro-2,4-dinitrobenzene for 4 h at pH 8 caused 86% inactivation, almost complete desensitization to allosteric inhibition by GTP, but only partial desensitization to ADP activation. The enzyme remained hexameric after such treatment. NAD⁺, but not NADH or NADPH, partially protected activity. Protection was enhanced by GTP and decreased by ADP. GTP and NADH together protected effectively, although separately neither protected. GTP and NADPH gave partial protection of activity. Glutarate and succinate, inhibitors competitive with glutamate, gave substantial protection, slightly enhanced in the presence of NAD⁺. With glutarate, but not succinate, an initial activation was seen during chemical modification. The allosteric response to GTP was protected by GTP itself only when NAD⁺ or NAD(P)H was also present; other ligands failed to protect. Similarly ADP alone did not protect ADP sensitivity. NADH partially protected ADP sensitivity, although NADPH did not. ADP itself counteracted the protection given by NADH. GTP with NADH completely protected ADP sensitivity. This combination of ligands thus protects all the assayed properties. GTP with NADPH gave less complete protection of the ADP response. Observed protection patterns varied with the pH and coenzyme concentration of the assay mixture under constant conditions of chemical modification. Overall, the results are inconsistent with the view that dinitrophenylation directly blocks nucleotide binding sites, and suggest rather that it interferes with communication between sites.     

Identification of hydrogen selenide and other volatile selenols by derivatization with 1-fluoro-2,4-dinitrobenzene

A procedure is described for the trapping and identification of hydrogen selenide and methyl selenol ( CH3SeH ). The volatile selenols were generated by reducing selenious acid or dimethyldiselenide with Zn dust and hydrochloric acid under a stream of nitrogen and passing into a trapping solution composed of 50 mM 1-fluoro-2,4-dinitrobenzene plus 83 mM sodium bicarbonate in 67% dimethylformamide:33% water. The selenols react rapidly to form stable dinitrophenyl (DNP) selenoethers that can be extracted into benzene; these are easily identified by TLC, HPLC, or mass spectrometry. Hydrogen selenide is trapped in 90-99% yield, primarily as the di-DNP- monoselenide with a trace of di-DNP- diselenide .