Modification Of Histidine Residues Research Articles

Chemical modifications of Serratia marcescens anthranilate synthase component I.

Serratia marcescens anthranilate synthase Component I (AS I) was purified from a plasmid-containing Escherichia coli strain. Residues essential for AS I function were studied by chemical modification reactions. Phenylglyoxal and 1,2-cyclohexanedione modified 2-5 arginine residues and inactivated AS I. The substrate chorismate reduced the rate of inactivation. Analysis of inactivation data indicated that 1 arginine residue is essential for activity. Histidine residues in AS I were modified by ethoxyformic anhydride and by photooxidation. Enzyme inactivation accompanied modification of histidine residues. Inactivation was prevented by substrate. Comparison of the number of carbethoxy groups incorporated between substrate-protected and unprotected AS I indicated that 1 histidine residue is required for activity. AS I was also inactivated by bromopyruvate. Substrate retarded inactivation by bromopyruvate. A differential labeling experiment indicated that the loss of AS I activity was correlated with alkylation of 1 cysteine residue. A tryptic peptide containing the essential cysteine residue was isolated. The peptide has the amino acid sequence of Ile-Cys-Gln-Ala-Gly-Ser-Arg.

The sulfhydryl groups of the thiol-dependent cytolytic toxin from Bacillus alvei evidence for one essential sulfhydryl group

Alveolysin, an extracellular protein toxin (M r ⋍ 63,000) excreted by Bacillus alvei and purified to homogeneity was shown to contain four cysteine residues. All thiol groups of the hemolytically active toxin preparation were free as found by direct titration by 5,5′-dithiobis (2-nitrobenzoic acid) and confirmed by the absence of disulfide bond. Toxin alkylation with tosyl lysine chloromethyl ketone resulted in the complete loss of hemolytic activity and the disappearance of only one thiol group with no modification of histidine residues. These results support the conclusion that one essential thiol group is implicated in the membrane-disrupting activity of alveolysin.

Inactivation of aspartyl proteinases by butane-2,3-dione. Modification of tryptophan and tyrosine residues and evidence against reaction of arginine residues

Butane-2,3-dione inactivates the aspartyl proteinases from Penicillium roqueforti and Penicillium caseicolum, as well as pig pepsin, penicillopepsin and Rhizopus pepsin, at pH 6.0 in the presence of light but not in the dark. The inactivation is due to a photosensitized modification of tryptophan and tyrosine residues. In the dark none of the amino acid residues, not even arginine residues, is modified even after several days. In the light one arginine residue in pig pepsin is lost at a rate that is comparable with the rate of inactivation; however, the loss of the single arginine residue in the aspartyl proteinase of P. roqueforti and the second arginine residue of pig pepsin is slower than the loss of activity; penicillopepsin is devoid of arginine. Loss of most of the activity is accompanied by the following amino acid losses: P. roqueforti aspartyl proteinase, about two tryptophan and six tyrosine residues; penicillopepsin, about two tryptophan and three tyrosine residues; pig pepsin, about four tryptophan and most of the tyrosine residues. Modification of histidine residues was too slow to contribute to inactivation. None of the other residues, including half-cystine and methionine residues (when present), was modified even after prolonged incubation. The inactivation of P. roqueforti aspartyl proteinase and pig pepsin appears due to non-specific modification of several residues. With penicillopepsin, however, the reaction is more limited and initially affects only those tryptophan and tyrosine residues that lie in the active-site groove. In the presence of pepstatin the rate of inactivation is considerably diminished. After prolonged reaction a general structural breakdown occurs.

Action pattern and active site of endo .BETA.-1,3-glucanase IV from Flavobacterium dormitator var. glucanolyticae FA-5.

The action pattern and amino acid residues involved in the active site of endo β-1, 3-glucanase IV from Flav. dormitator var. glucanolyticae FA-5 were investigated. The glucanase was specific for straight chains of β-1, 3-glucosidic linkages. The enzyme endowisely hydrolyzed β-1, 3-ghicans to produce laminaripentaose and showed transglucanosylation activity. The glucanase was inactivated by modification of histidine residues with diethylpyrocarbonate and by photooxidation, and by modification of carboxyl residues with Woodward's reagent K and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. The presence of β-1, 3-ghicans resulted in protection of the enzyme from inactivation by modifications, suggesting that these residues are at the β-1, 3- glucan binding and/or active site of the glucanase molecule.

Studies on the purification and structure-function relationships of murine lymphocyte activating factor (interleukin 1)

Lymphocyte activating factor [LAF; Interleukin 1 (IL 1)] obtained from the murine macrophage cell line, P388D 1, was partially purified by ammonium sulfate fractionation and chromatography on DEAE cellulose, Sephacryl S200 and phenyl Sepharose. Relative to the starting material (sp. act. = 17 U/mg protein), the LAF (IL 1) at the phenyl Sepharose stage of purification was 5–10% pure (sp. act. = 2 × 10 4 U/mg protein) with a yield of 7–30%, depending on the volume of starting material. Analysis of the LAF (IL 1) by isoelectrofocusing in polyacrylamide gels revealed that the LAF (IL 1) obtained after elution from the phenyl Sepharose column was associated specifically with one of eight bands stained with Coomassie Brilliant Blue. A specific activity of ≥3 × 10 5 U/ mg protein was estimated for a homogeneous preparation of LAF (IL 1). LAF (IL 1) activity was lost after modification of arginine residues by phenylglyoxal and was only weakly affected by modification of histidine residues with diethylpyrocarbonate. No active fragments were obtained after cyanogen bromide cleavage of LAF (IL 1).

Isolation of τ-phosphohistidine from a phosphoryl-enzyme intermediate of human prostatic acid phosphatase

The carbethoxylation of prostatic acid phosphatase (orthophosphoricmonoester phosphohydrolase (acid optimum), EC 3.1.3.2) was accompanied by modification of histidine residues and the inactivation of the enzyme. These findings are consistent with photoinactivation experiments described earlier (Rybarska, J. and Ostrowski, W. (1974) Acta Biochim. Polon. 21, 377–390). Prostatic acid phosphatase was phosphorylated at alkaline pH using p-nitrophenyl [ 32P]phosphate as substrate. Phosphoryl enzyme is stable solutions and undergoes dephosphorylation at acidic pH. After hydrolysis of phosphoryl enzyme in strong alkaline solution, a single phosphoryl amino acid was isolated from hydrolyzate and identified as the τ-phosphohistidine.

Photooxidation of fibrinogen in the presence of methylene blue and its effect on polymerization

Human fibrinogen was illuminated in the presence of methylene blue. The resulting photooxidized fibrinogen was devoid of polymerization activity and thrombin-induced coagulability. The initial rate of the thrombin catalysed release of fibrinopeptides from photooxidized fibrinogen was normal. It was shown that illumination of photooxidized fibrinogen and photooxidized fragment N-DSK caused the modification of histidine residues. Tryptophan residues were also modified. When fibrinogen was photooxidized immediately after the addition of thrombin, the capacity to polymerize was lost. The inhibition of polymerization was less marked when oxidation was initiated at the time when polymerization began or thereafter. Photooxidized fibrinogen acts as an inhibitor of the polymerization of fibrin monomers. Photooxidized fibrinogen has affinity for thrombin-activated fibrinogen-Sepharose and thrombin-activated fragment N-DSK-Sepharose. When the former conjugate is illuminated in the presence of methylene blue its affinity for fibrinogen is decreased. It is concluded that the fragment N-DSK domain of fibrinogen is affected by photooxidation.

The structure and function of ribonuclease T1. XXI. Modification of histidine residues in ribonuclease T1 with iodoacetamide.

1. When ribonuclease T1 [EC 3.1.4.8] (0.125% solution) was treated with a 760-fold molar excess of iodoacetamide at pH 8.0 and 37 degrees, about 90% of the original activity was lost in 24 hr. The half-life of the activity was about 8 hr. The binding ability for 3'-GMP was lost simultaneously. Changes were detected only in histidine and the amino-terminal alanine residues upon amino acid analyses of the inactivated protein and its chymotryptic peptides. The inactivation occurred almost in parallel with the loss of two histidine residues in the enzyme. The pH dependences of the rate of inactivation and that of loss of histidine residues were similar and indicated the implication of a histidine residue or residues with pKa 7.5 to 8 in this reaction. 3'-GMP and guanosine showed some protective effect against loss of activity and of histidine residues. The reactivity of histidine residues was also reduced by prior modification of glutamic acid-58 with iodoacetate, of lysine-41 with maleic or cis-aconitic anhydride or 2,4,6-trinitrobenzenesulfonate or of arginine-77 with ninhydrin. 2. Analyses of the chymotryptic peptides from oxidized samples of the iodoacetamide-inactivated enzyme showed that histidine-92 and histidine-40 reacted with iodoacetamide most rapidly and at similar rates, whereas histidine-27 was least reactive. Alkylation of histidine-92 was markedly slowed down when the Glu58-carboxymethylated enzyme was treated with iodoacetamide. On the other hand, alkylation of histidine-40 was slowed down most in the presence of 3'-GMP. These results suggest that histidine-92 and histidine-40 are involved in the catalytic action, probably forming part of the catalytic site and part of the binding site, respectively, and that histidine-27 is partially buried in the enzyme molecule or interacts strongly with some other residue, thus becoming relatively unreactive.

Chemical modifications of histidine residues in cytoplasmic asparate aminotransferase from beef kidney.

Holo and apoenzyme of aspartate aminotransferase from beef kidney are 80% inactivated by photoxidation in the presence of 2 X 10(-6) M tetraiodofluroescein with the modification of two histidine residues per enzyme protomer. At a higher concentration (1 X 10(-5) M) a tyrosine residue is also modified. The keto substrates, ketoglutarate and oxalacetate, protect the enzyme from photoxidation. Diethylpyrocarbonate modifies three histidine residues per enzyme protomer and reduces the activity only 10%. These results suggest that the two histidine residues photoxidized through the sensitizer, are located in the active site of the enzyme, at least one of these appears to be involved in ketosubstrate binding. The other three histidines modified by diethylpyrocarbonate are likely located on the enzyme surface and are not involved in the catalytic activity of the enzyme.

Inhibition of the self-assembly of tubulin by diethylpyrocarbonate and photooxidation

Chemical modification of tubulin by photooxidation and by reaction with diethylpyrocarbonate inhibits the in vitro formation of microtubules. This inhibition apparently results from the modification of histidine residues, since the inhibition by diethylpyrocarbonate is reversed by hydroxylamine and the pH dependence of the rate of photooxidation shows the involvement of a group with a pKa value of about 6.5. The inhibition of self-assembly results from the modification of not more than three histidine residues. Sulfhydryl residues are not modified under the experimental conditions used. Colchicine and GTP binding by tubulin were not greatly affected under conditions which completely inhibited the polymerization.

Modification of histidine residues in yeast pyruvate kinase by diethylpyrocarbonate.

Modification of histidine residues in yeast pyruvate kinase by diethylpyrocarbonate.

Synthetic thyrotropin-releasing factor analogs. 3. Effect of replacement or modification of histidine residue on biological activity.

Synthetic thyrotropin-releasing factor analogs. 3. Effect of replacement or modification of histidine residue on biological activity.

Effect of Alkylation of Sperm Whale Myoglobin on Response to Extremes of Temperature and pH

Abstract Carboxymethyl and carboxamidomethyl derivatives of sperm whale ferrimyoglobin were prepared by reaction near pH 6.7 with bromoacetate and iodoacetamide, respectively. As had been established earlier, unless the reaction was carried to an extreme stage in the modification of histidine residues, the derivatives obtained were stable at neutral pH. The derivatives were generally less stable than the unreacted protein at high pH values, as judged by measurements of absorption and optical rotatory spectra. Of the two types of derivatives, the less highly negatively charged carboxamidomethyl myoglobin proved the more stable at pH 11.5 and above. In acid solution at pH 3.2 or pH 2.2, the derivatives, like the unreacted protein, showed a characteristically fully altered absorption spectrum. Optical rotatory measurements showed, however, that the apparent helix content decreased between pH 3.2 and pH 2.2. The decrease was least marked with the less strongly positively charged carboxymethyl myoglobin preparations. Both types of derivative behaved much like the unreacted protein in experiments in which the optical rotation at 233 mµ was studied as a function of temperature up to 80° or 85°. At these highest temperatures, the apparent order of stability was unreacted myoglobin g carboxamidomethyl derivative g carboxymethyl derivative, and aggregation with precipitation developed in a few minutes. The unreacted protein showed little or no tendency to redissolve on recooling to 23°. However, both derivatives were recovered to the extent of about 60% as apparently native protein. It was concluded that although electrostatic factors probably played a discernible role in determining the relative stabilities of the proteins at extremes of pH, such factors were not prominent in affecting the conformational transitions at high temperatures. Either modified derivative proved much better able to recover the native conformation on cooling than the unreacted protein. It is suggested that the advantage in having the external histidine residues modified may lie in the coding introduced to specify that such residues shall remain at all times in a hydrophilic environment, and not become trapped in erroneous, internal conformational variants.

MODIFICATION OF HISTIDINE RESIDUES LEADING TO THE APPEARANCE OF VISIBLE FLUORESCENCE.

The object of this investigation was to study the modification of histidine residues by N -bromosuccinimide leading to the appearance of visible fluorescence. 1. 1. Histidine and tryptophan yield visible fluorescence when treated with N -bromosuccinimide in phosphate buffer at neutral pH. Under the conditions required for the formation of the fluorescent products, tryptophan and tyrosine lose their native ultraviolet fluorescence. 2. 2. Details are presented in peptides yield mmore fluorescence than histidine itself. The amount of fluorescence formed differs from one peptide to another depending on the amino acids adjacent to the histidine residue. 3. 4. The reaction leading to the formation of fluorescent products from histidine residues can experimentally be divided into two distinct steps: (a) Rapid attack of the histidine residue by N -bromosuccinimide to yield a non-fluorescent intermediate. (b) Conversion of the intermediate into a fluorescent compound in a slow reaction cataylsed by phosphate (or pyrophosphate, or hydroxyl ions). Optimal reaction conditions for both steps were determined. 4. 5. The formation of fluorescent products from histidine residues when treated with N -bromosuccinimide may be of use in following the modification of histidine residues in peptides and in proteins. It may also be a means of introducing visible fluorescence into proteins for structural studies. 5. 6. The possible relevance of this work to the problem of oxidative phosphorylation is briefly discussed.