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PubMed1977

Chemical modification of cellulase from Aspergillus niger.

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.

Aspergillus niger↗
PubMed1999

Chemical modification and site-directed mutagenesis of human liver arginase: evidence that the imidazole group of histidine-141 is not involved in substrate binding.

Native and wild-type recombinant human liver arginases (EC 3.5.3.1) were photoinactivated by Rose bengal, and protection was afforded by the competitive inhibitor l-lysine. The dissociation constant for the enzyme-protector complex was essentially equal to the corresponding K(i) value. Upon mutation of His141 by phenylalanine, the enzyme activity was reduced to 6-10% of wild-type activity, with no changes in K(m) for arginine or K(i) for l-lysine or l-ornithine. The subunit composition of active enzyme was not altered by mutation, but the mutant H141F was markedly more sensitive to trypsin inactivation and completely insensitive to inactivation by diethyl pyrocarbonate (DEPC) and photoinactivation. Species with histidine groups blocked with DEPC were also insensitive to photoinactivation. We conclude that His141, which is the target for both inactivating procedures, is not involved in substrate binding, but plays a critical, albeit not essential role in the hydrolysis of enzyme-bound substrate.

Arginase↗
PubMed1994

Involvement of essential histidine residue(s) in the activity of Ehrlich cell plasma membrane NADH-ferricyanide oxidoreductase.

The existence of histidine residue(s) implicated in the active site of NADH-ferricyanide oxidoreductase in plasma membrane vesicles isolated from Ehrlich ascites tumour cells is investigated. The shape of the pH-dependence curve of the enzyme activity suggests that one or more histidine residues are located at (or near) the active site of the enzyme. This hypothesis is supported by the following experimental data: the loss of activity after treatment with diethyl pyrocarbonate (DEPC) or photooxidation by using Rose bengal, and the strong inhibition caused by Zn2+ ions at micromolar concentrations. The combined arguments support the statement that histidine plays an essential role in the catalytic activity of NADH-ferricyanide oxidoreductase from Ehrlich ascites tumour cells.

Animals↗
PubMed1991

RNA recognition by Tat-derived peptides: interaction in the major groove?

Replication of human immunodeficiency virus requires binding of the viral Tat protein to its RNA target sequence TAR; peptides derived from Tat bind to a TAR "contact site" spanning 5 bp and a trinucleotide pyrimidine bulge. We find that high affinity binding requires a U residue in the bulge loop and 2 specific adjacent base pairs. Other bulged RNAs bind in a lower affinity nonspecific manner; sequence-specific binding requires a bulge loop of more than 1 nucleotide. Reaction with diethyl pyrocarbonate indicates that one effect of the bulge is to make the otherwise deep and narrow RNA major groove accessible. A model consistent with these data involves local distortion of A-form geometry at the bulge, which bends the helix and permits protein binding and interactive access in the RNA major groove.

Amino Acid Sequence↗
PubMed1999

Contribution of histidine residues to oligomerization of theta-toxin (perfringolysin O), a cholesterol-binding cytolysin.

Theta-toxin (perfringolysin O) modified by diethyl pyrocarbonate, a histidine-specific reagent, lost its hemolytic activity. The modified toxin retains the activities of binding to and insertion into cholesterol-containing membranes but lacks the ability to form oligomers. These results suggest that histidine residues of theta-toxin contribute their share to cytolysis, especially the oligomerization process.

Bacterial Toxins↗
PubMed1975

Interactions of urdine diphosphate glucose dehydrogenase with the inhibitor urdine diphosphate xylose.

1. UDP-xylose and UDP-glucose both bind to UDP-glucose dehydrogenase in the absence of NAD+, causing an enhancement of protein fluorescence. 2. The binding of UDP-xylose is pH-dependent, tighter binding being observed at pH8.2 than at pH8.7. 3. At low protein concentrations sigmiodal profiles of fluorescence enhancement are obtained on titration of the enzyme with UDP-xylose. As the protein concentration is increased the titration profiles become progressively more hypebolic in shape. 4. The markedly different titration profiles obtained on titrating enzyme and the enzyme-NAD+ complex with UDP-xylose suggests a conformational difference between these two species 5. NAD+ lowere the apparent affinity of the enzyme for UDP-xylose. 6. There is no change in the apparent moleculare weight of UDP-glucose dehydrogenase on binging UDP-xylose. 7. Protein modification by either diethyl pyrocarbonate or 5, 5'-dithiobis-(2-nitrobenzoate) does not "desensitize" the enzyme with respect to the inhibition by UDP-xylose. 8. UDP-xylose lowers the affinity of the enzyme for NADG. 9. It is suggested that UDP-xylose is acting as a substrate analogue of UDP-glucose and causes protein-conformational changes on binding to the enzyme.

Alcohol Oxidoreductases↗
PubMed1999

Functional characteristics of basolateral peptide transporter in the human intestinal cell line Caco-2.

The apical H+-coupled peptide transporter (PEPT1) and basolateral peptide transporter in human intestinal Caco-2 cells were functionally compared by the characterization of [14C]glycylsarcosine transport. The glycylsarcosine uptake via the basolateral peptide transporter was less sensitive to medium pH than uptake via PEPT1 and was not transported against the concentration gradient. Kinetic analysis indicated that glycylsarcosine uptake across the basolateral membranes was apparently mediated by a single peptide transporter. Small peptides and beta-lactam antibiotics inhibited glycylsarcosine uptake by the basolateral peptide transporter, and these inhibitions were revealed to be competitive. Comparison of the inhibition constant values of various beta-lactam antibiotics between PEPT1 and the basolateral peptide transporter suggested that the former had a higher affinity than the latter. A histidine residue modifier, diethyl pyrocarbonate, inhibited glycylsarcosine uptake by both transporters, although the inhibitory effect was greater on PEPT1. These findings suggest that a single facilitative peptide transporter is expressed at the basolateral membranes of Caco-2 cells and that PEPT1 and the basolateral peptide transporter cooperate in the efficient transepithelial transport of small peptides and peptidelike drugs.

Caco-2 Cells↗
PubMed2004

Purification, tandem mass characterization, and inhibition studies of oxidosqualene-lanosterol cyclase enzyme from bovine liver.

The oxidosqualene-lanosterol cyclase (OSC) from bovine liver has been isolated from the microsomal membrane fraction and purified to homogeneity by ultracentrifugation, Q-Sepharose, hydroxyapatite, and HiTrap heparin chromatographies. The purified protein required Triton X-100 to retain its highest activity. The cyclase had a molecular mass of approximately 70 and approximately 140 kDa, as evidenced by a single protein band on silver-stained SDS-PAGE and Coomassie-stained PAGE, respectively. Results from Edman degradation of OSC suggested that it might have a blocked N-terminus. Further peptide mapping coupled with tandem mass spectrometric determination identified three peptide fragments, ILGVGPDDPDLVR, LSAEEGPLVQSLR, and NPDGGFATYETK, which are highly homologous to human, rat, and mouse OSCs. The purified cyclase showed pH and temperature optima at pH 7.4 and 37 degrees C, respectively. The apparent K(M) and k(cat)/K(M) values were estimated to be 11 microM and 1.45 mM(-1)min(-1), respectively. Inhibition studies using both Ro48-8071 and N-(4-methylenebenzophenonyl)pyridinium bromide showed potent inhibition of OSC with an IC(50) of 11 nM and 0.79 microM, respectively. Results from DTNB modification and DTNB coupled with Ro48-8071 competition study suggest that two sulfhydryl groups are involved in the catalysis but not located in the substrate binding pocket or catalytic active site. The purified OSC was maximally inactivated by diethyl pyrocarbonate near neutral pH and re-activated by hydroxylamine, indicating the modification of histidine residues. The stoichiometry of histidine modification and the extent of inactivation showed that two essential histidine residues per active site are necessary for complete bovine liver OSC activity.

Amino Acid Sequence↗
PubMed1988

[Localization of melted regions in supercoiled DNA by means of chemical modification].

Diethyl pyrocarbonate was used as a probe in mapping early melting stages in supercoiled DNA. It was shown that in the process of early melting of pAO3 DNA two denatured regions (about 15 b.p.) arouse near the left and right boundaries of the cruciform structure. In course of further melting denatured regions appeared within AT-rich stretches and the cruciform structure itself disappeared.

DNA, Superhelical↗
PubMed1985

Acid-base catalysis in the argininosuccinate lyase reaction.

The pH variation of the kinetic parameters, Vmax and V/K, was examined for the forward and reverse reaction of bovine liver argininosuccinate lyase. In the forward reaction the Vmax profile showed one group that must be unprotonated for activity over the pH range 5-10. The V/K profile for argininosuccinate showed one group that must be unprotonated and two groups that must be protonated for activity. The Vmax profile for the reverse reaction showed only one group that must be protonated for activity. These results support the proposal that catalysis is facilitated in the forward reaction by a general base that abstracts a proton from C-3 of argininosuccinate and a general acid that donates a proton to the guanidinium nitrogen during carbon-nitrogen bond cleavage. The enzyme is completely inactivated by diethyl pyrocarbonate or a water-soluble carbodiimide at pH 6. These experiments suggest that a histidine and a carboxyl group are at or near the active site and are essential for catalytic activity. The observed shifts of the pH profiles of the forward reaction with temperature and organic solvent (25% dioxane) were also consistent with a histidine and carboxylate group.

Animals↗
PubMed1976

Purification and characterisation of polygalacturonases from a commercial Aspergillus niger preparation.

The polygalacturonase (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) activity of Pectinol is resolved into two fractions (E1 and E2) of about equal total activity on DEAE-cellulose. These fractions are purified from other pectinolytic enzyme activity by Sephadex G-75 chromatography. Both E1 and E2 reduce the viscosity of polygalacturonate by 50% after 7% of the glycosidic bonds are hydrolysed. Their activities are not affected by iodoacetate (1 mM) or EDTA (10 mM). E1 and E2 have different molecular weights (35 000 and 85 000, respectively) and different electrophoretic mobilities on sodium dodecyl sulphate polyacrylamide gels. Their pH (4.1 and 3.8 respectively) and ionic strength optima and specific activities also differ. Both enzymes are inhibited at similar rates by diethyl pyrocarbonate at pH 6 but only E2 is protected from this inhibition by 2% (w/v) polygalacturonate. The rate of change of protein absorbance at 250 nm accompanying this inhibition, and the residues are essential for the activities of both E1 and E2. About 2 molecules of carbethoxyhistidine per subunit of E2 and 0.6 molecules per subunit of E1 are present in the completely inhibited enzymes.20

Aspergillus↗
PubMed1989

Ecori DNA methylase activity is eliminated upon histidine residue modification.

The E. coli EcoRI DNA methylase activity is completely eliminated in five minutes upon incubation with the histidine residue specific reagent diethyl pyrocarbonate. In that two moles of N-ethoxyformylimidazole per mole of methylase are detected spectroscopically upon inactivation and activity is not restored by hydroxylamine, it is likely that activity loss is due to double modification of a single histidine residue. This information is critical in determining the enzymatic mechanism, causes of the pH-activity curve, designing protein mutants and interpreting previous structure-function data.

Chemical Phenomena↗
PubMed1992

The role of histidine 63 in the catalytic mechanism of Bordetella pertussis adenylate cyclase.

Of the 9 histidines located in the catalytic domain of Bordetella pertussis adenylate cyclase, three (His63, His106, and His298) were found to be conserved in the adenylate cyclase of Bacillus anthracis, another calmodulin-dependent enzyme. Substitution of His63 with Arg, Glu, Gln, or Val decreased the catalytic efficiency of adenylate cyclase between 2 and 3 orders of magnitude and altered the kinetic properties of the enzyme. These effects varied in relation to the nature of the substituting residue, pH, and direction of the reaction, i.e. ATP cyclization (forward) or ATP synthesis (reverse). Arg was the best substituent for His63 as catalyst in the forward reaction, with shift of the optimum pH to the alkaline side, whereas Glu was the best substituent for His63 in the reverse reaction, with shift of the optimum pH to the acidic side. Diethyl pyrocarbonate, which had a deleterious effect on wild-type adenylate cyclase was ineffective on His63 mutants. From these results we conclude that His63 is involved in the reaction mechanism of adenylate cyclase, which requires a general acid/base catalyst, most probably as an intermediate in a charge-relay system.

Adenylyl Cyclases↗
PubMed1997

Histidine modification of human serum butyrylcholinesterase.

The effects of histidine-modifying reagents on human serum butyrylcholinesterase (BChE) were investigated. The commercially available enzyme was further purified by chromatography on a Sepharose CI-6B column prior to use. In the modification studies, we found that the histidine-specific reagents tosylphenylalanine chloromethyl ketone (TPCK) and tosyllysine chloromethyl ketone (TLCE) did not modify the enzyme; however, they inhibited the enzyme reversibly. The kinetic parameters of enzyme inhibition calculated were alpha = 10.8, beta = 0.26, and Ki = 0.016 mM for TPCK. TLCK inhibition gave similar kinetic behavior, with alpha = 41.6, beta = 0.065, and Ki = 0.039 mM. Tosyllysine, an analog of TLCK, did not inhibit the enzyme. Removal of TPCK and TLCK by dialysis resulted in significant reactivation of the enzyme. From kinetic studies, it was found that the inhibitions were hyperbolic mixed-type inhibitions. We concluded that the reagents competed with substrate for hydrophobic binding sites and inhibited the enzyme reversibly. On the other hand, in the modification studies with diethyl pyrocarbonate (DPC), it was observed that inactivation of the enzyme was irreversible and time-dependent. In the protection studies, the activity of the enzyme was partially protected from inactivation by DPC even at a 50 mM concentration of butyrylthiocholine. The results indicate that DPC modifies some essential histidine side chains in BChE, including the functional histidyl residue found at the active site.

Binding Sites↗
PubMed2006

Characterization of a thermostable carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima.

The gene encoding carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima (tm0053) was cloned. The recombinant protein (EST53) was overexpressed in Escherichia coli without its NH(2)-terminal hydrophobic region, and with a C-terminal hexahistidine sequence. The enzyme was purified to homogeneity by heat treatment, followed by Ni(2+) affinity chromatography, and then characterized. Among the p-nitrophenyl esters tested, the best substrate was p-nitrophenyl decanoate with K (m) and k (cat) values of 3.1 muM and 10.8 s(-1), respectively, at 60 degrees C and pH 7.5. The addition of O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid decreased the esterase activity, indicating that EST53 is dependent on the presence of Ca(2+) ion. In addition, its activity was inhibited by the addition of phenylmethylsulfonyl fluoride and diethyl pyrocarbonate, indicating that it contains serine and histidine residues, which play key roles in the catalytic mechanism. EST53 shows a relatively high degree of similarity to Burkholderia lipases that belong to family I.2 of the lipolytic enzymes, whereas the local sequence around the pentapeptide of EST53 is most similar to those of Bacillus lipases belonging to family I.4.

Amino Acid Sequence↗
PubMed1986

Identification of amino acid residues essential for enzyme activity of sheep liver 5,10-methylenetetrahydrofolate reductase.

Sheep liver 5,10-methylenetetrahydrofolate reductase was subjected to specific chemical modification with phenylglyoxal, diethyl pyrocarbonate and N-bromosuccinimide. The second-order rate constants for inactivation were calculated to be 54 M-1 X min-1, 103 M-1 X min-1 and 154 M-1 X min-1 respectively. This inactivation could be prevented by incubation with substrates or products, suggesting that the residues modified, namely arginine, histidine and tryptophan, are essential for enzyme activity.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗
PubMed1994

Efficient independent activity of a monomeric, monofunctional dehydroquinate synthase derived from the N-terminus of the pentafunctional AROM protein of Aspergillus nidulans.

The dehydroquinate synthase (DHQ synthase) functional domain from the pentafunctional AROM protein of Aspergillus nidulans has previously been overproduced in Escherichia coli [van den Hombergh, Moore, Charles and Hawkins (1992) Biochem J. 284, 861-867]. We now report the purification of this domain to homogeneity and subsequent characterization. The monofunctional DHQ synthase was found to retain efficient catalytic activity when compared with the intact pentafunctional AROM protein of Neurospora crassa [Lambert, Boocock and Coggins (1985) Biochem J. 226, 817-829]. The apparent kcat. was estimated to be 8 s-1, and the apparent Km values for NAD+ and 3-deoxy-D-arabino-heptulosonate phosphate (DAHP) were 3 microM and 2.2 microM respectively. These values are similar to those reported for the intact N. crassa enzyme, except that the apparent Km for NAD+ reported here is 15-fold higher. The monofunctional DHQ synthase domain is inactivated by treatment with chelating agents in the absence of substrates and is re-activated by the addition of metal ions; among those tested, Zn2+ gave the highest kcat./Km value. The enzyme is inactivated by diethyl pyrocarbonate; both the substrate, DAHP, and the product phosphate protected against inactivation. Size-exclusion chromatography suggested an M(r) of 43,000 for the monofunctional domain, indicating that it is monomeric and compactly folded. The c.d. spectrum confirmed that the domain has a compact globular conformation; the near-u.v. c.d. of zinc- and cobalt-reactivated domains were superimposable.

Alcohol Oxidoreductases↗
PubMed1992

Pseudomonas aeruginosa exotoxin A interaction with eucaryotic elongation factor 2. Role of the His426 residue.

Pseudomonas aeruginosa exotoxin A (ETA) catalyzes the transfer of the ADP-ribose moiety of NAD+ onto eucaryotic elongation factor 2 (EF-2). To study the ETA site of interaction with EF-2, an immobilized EF-2 binding assay was developed. This assay demonstrates that ETA, in the presence of NAD+, binds to immobilized EF-2. Additionally, diphtheria toxin was also found to bind to the immobilized EF-2 in the presence of NAD+. Comparative analysis was performed with a mutated form of ETA (CRM 66) in which a histidine residue at position 426 has been replaced with a tyrosine residue. This immunologically cross-reactive, ADP-ribosyl transferase-deficient toxin does not bind to immobilized EF-2, thus explaining its lack of ADPRT activity. ETA bound to immobilized EF-2 cannot bind the monoclonal antibody TC-1 which specifically recognizes the ETA epitope containing His426. Immunoprecipitation of native ETA by mAb TC-1 is only achieved by incubating ETA in the presence of NAD+. Diethyl pyrocarbonate modification of the His426 residue blocks ETA binding to EF-2 and prevents the binding of the TC-1 antibody. Analogs of NAD+ containing a reduced nicotinamide ring or modified adenine moieties cannot substitute for NAD+ in the immobilized binding assay. Collectively, these data support our proposal that the site of ETA interaction with EF-2 includes His426 and that a molecule of NAD+ is required for stable interaction.

ADP Ribose Transferases↗