Chemical modification of chloroperoxidase with diethylpyrocarbonate. Evidence for the presence of an essential histidine residue

Chloroperoxidase from Caldariomyces fumago is well documented as an extremely versatile catalyst, and studies are currently being conducted to delineate the fine structural features that allow the enzyme to possess chemical and physical similarities to the peroxidases, catalases, and P-450 cytochromes. Earlier investigations of ligand binding to the heme iron of chloroperoxidase, along with the presence of an invariant distal histidine residue in the active site of peroxidases and catalases, have led to the hypothesis that chloroperoxidase also possesses an essential histidine residue that may participate in catalysis. To address this in a more direct fashion, chemical modification studies were initiated with diethylpyrocarbonate. Incubation of chloroperoxidase with this reagent resulted in a time-dependent inactivation of enzyme. Kinetic analysis revealed that the inactivation was due to a simple bimolecular reaction. The rate of inactivation exhibited a pH dependence, indicating that modification of a titratable residue with a pKa value of 6.91 was responsible for inactivation; this data provided strong evidence for histidine derivatization by diethylpyrocarbonate. To further support these results, inactivation due to cysteine, tyrosine, or lysine modification was ruled out. The stoichiometry of histidine modification was estimated by the increase in absorption at 246 nm, and it was found that more than 1 histidine residue was derivatized when chloroperoxidase was inactivated with diethylpyrocarbonate. However, it was shown that the rates of modification and inactivation were not equivalent. This was interpreted to reflect that both essential and nonessential histidine residues were modified by diethylpyrocarbonate. Kinetic analysis indicated that modification of a single essential histidine residue was responsible for inactivation of the enzyme. Studies with [14C]diethylpyrocarbonate provided stoichiometric support that derivatization of a single histidine inactivated chloroperoxidase. Based on sequence homology with cytochrome c peroxidase, histidine 38 was identified as a likely candidate for the distal residue. Molecular modeling, based on secondary structure predictions, allows for the construction of an active site peptide, and implicates a number of other residues that may participate in catalysis.     

Evidence for histidine residues in the immunoglobulin-binding site of human C1q

The immunoglobulin-binding activity of subcomponent Clq of human complement is lost following treatment with diethylpyrocarbonate; the inactivation showed first-order kinetics with respect to time and modifier concentration. Soluble IgG oligomers protected Clq against diethylpyrocarbonate modification. Treatment of modified Clq with hydroxylamine resulted in an 85% recovery of its ability to bind to aggregated immunoglobulin. The inactivation process was associated with modification of 12.1 +/- 0.7 histidine residues per Clq molecule. These data are consistent with the presence of histidine residues in the immunoglobulin-binding sites of Clq; these residues may participate in ionic interactions with the carboxyl groups known to be in the Clq binding site of IgG.     

Inactivation of a beta-glucosidase from Arthrobotrys conoides by diethyl pyrocarbonate: evidence of histidine at the active site.

Modification of A. conoides beta-glucosidase by diethylpyrocarbonate caused rapid inactivation of the enzyme. The kinetic analyses showed that the inactivation by diethylpyrocarbonate resulted from the modification of an average of one histidine residue per mole of enzyme. The modified enzyme showed an increase in absorbance at 240 nm. Sulphydryl, lysine and tyrosine residues were not modified by diethylpyrocarbonate treatment. The substrate offered significant protection against diethylpyrocarbonates modification. The results indicate that diethylpyrocarbonate was interacting with the enzyme at or near the active site.     

Evidence for the participation of histidine residues located in the 56 kDa C-terminal polypeptide domain of ADP-ribosyl transferase in its catalytic activity

Purified ADPRT protein was inactivated by the histidine specific reagent diethylpyrocarbonate, binding to two histidine residues, or by a relatively histidine selective photoinactivation method. Inactivation with up to 1.3 mM diethylpyrocarbonate was reversible by hydroxylamine. Enzymatic inactivation coincided with the loss of binding capacity of the enzyme protein to benzamide affinity matrix but not to DNA cellulose. Labelled diethylpyrocarbonate was identified exclusively in the 56 kDa carboxyl-terminal polypeptide where 2 out of 13 histidine residues were modified by this reagent. It is proposed that histidine residues in the 56 kDa polypeptide may participate as initiator sites for polyADP-ribosylation.     

Essential histidine residues in lysolecithin:lysolecithin acetyltransferase from rabbit lung

Both activities of rabbit lung lysolecithin:lysolecithin acyltransferase (EC 3.1.1.5), hydrolysis and transacylation, are inactivated by diethylpyrocarbonate. The reaction follows pseudo-first-order kinetics, and second-order rate constants of 1.17 mM-1min-1 for hydrolysis and 0.56 mM-1 min-1 for transacylation were obtained at pH 6.5 and 37 degrees C. The rate of inactivation is dependent on pH, showing the involvement of a group with a pK of 6.5. The difference spectra showed an increase in absorbance at 242 nm, indicating the modification of histidine residues. The activity lost by diethylpyrocarbonate modification can be partially recovered by hydroxylamine treatment. The statistical analysis of residual fractional activity versus the number of modified histidine residues leads to the conclusion that two histidine residues are essential for the hydrolytic activity, whereas transacylation activity depends on only one essential histidine. The substrate and substrate analogs protected the enzyme against inactivation by diethylpyrocarbonate, suggesting that the essential residues are located at or near the active site of the enzyme.     

Possible role of histidine in the L-proline transport system of Saccharomyces cerevisiae

The L-proline transport system of Saccharomyces cerevisiae is shown to be specifically inactivated upon incubation of intact yeast cells with the histidine modifier diethylpyrocarbonate. The extent of inactivation is half-maximum at 0.5 mM diethylpyrocarbonate for an incubation of 2 min at 30 degrees C and pH 6.0. Under the same conditions, the time dependence of inactivation is monophasic with the second-order rate constant of 5.5 M-1 X s-1 and the maximum rate Jmax of L-proline transport is lowered by about 50%, while the KT value remains unchanged. Moreover, L-proline afforded significant protection against diethylpyrocarbonate inactivation. The complete reactivation of a partially inactivated L-proline transport system by neutral hydroxylamine and the elimination of the possibility that the modification of other amino acid residues are responsible for the inactivation, suggested that the transport protein inactivation occurs solely by a modification of histidine residues.     

Chemical modification by diethylpyrocarbonate of an essential histidine residue in 3-ketovalidoxylamine A C-N lyase

3-Ketovalidoxylamine A C-N lyase of Flavobacterium saccharophilum is a monomeric protein with a molecular weight of 36,000. Amino acid analysis revealed that the enzyme contains 5 histidine residues and no cysteine residue. The enzyme was inactivated by diethylpyrocarbonate (DEP) following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with hydroxylamine, the enzyme activity was completely restored. The difference absorption spectrum of the modified versus native enzyme exhibited a prominent peak around 240 nm, but there was no absorbance change above 270 nm. The pH-dependence of inactivation suggested the involvement of an amino acid residue having a pKa of 6.8. These results indicate that the inactivation is due to the modification of histidine residues. Substrates of the lyase, p-nitrophenyl-3-ketovalidamine, p-nitrophenyl-alpha-D-3-ketoglucoside, and methyl-alpha-D-3-ketoglucoside, protected the enzyme against the inactivation, suggesting that the modification occurred at or near the active site. Although several histidine residues were modified by DEP, a plot of log (reciprocal of the half-time of inactivation) versus log (concentration of DEP) suggested that one histidine residue has an essential role in catalysis.     

The effect of histidine modification on the activity of myo-inositol monophosphatase from bovine brain.

The pH dependence of myo-inositol monophosphatase may indicate a role for histidine residues in the catalytic mechanism (Ganzhorn, A. J., and Chanal, M.-C. (1990) Biochemistry 29, 6065-6071). This possibility was investigated by chemical modification. At pH 6.0 and 25 degrees C, the enzyme was inactivated by diethylpyrocarbonate in a pseudo-first order reaction with a bimolecular rate constant of 0.37 M-1 s-1. Two histidines were modified rapidly with no effect on enzyme activity, while 3 residues were modified at a slower rate corresponding to the rate of inactivation. No noticeable changes in the secondary structure of the enzyme were observed by comparison of circular dichroic spectra before and after modification. Treatment of myo-inositol monophosphatase with diethylpyrocarbonate in the presence of inositol 1-phosphate, Mg2+, and Li+ protected 2 residues from modification and decreased the inactivation rate by about 5-fold. Spectrophotometric analysis, the restoration of enzyme activity by hydroxylamine, and the lack of any inhibitory effect with alkylating agents suggest that inactivation is due solely to modification of histidine. We conclude that a histidine residue is essential for activity and may act as a base catalyst during hydrolysis of the substrate.     

Chemical modification of sheep-liver 6-phosphogluconate dehydrogenase by diethylpyrocarbonate. Evidence for an essential histidine residue

Sheep liver 6-phosphogluconate dehydrogenase is shown to be inactivated by diethylpyrocarbonate in a biphasic manner at pH 6.0, 25 degrees C. After allowing for the hydrolysis of the reagent, rate constants of 56 M-1 s-1 and 11.0 M-1 s-1 were estimated for the two processes. The complete reactivation of partially inactivated enzyme by neutral hydroxylamine, the elimination of the possibility that modification of cysteine or tyrosine residues are responsible for inactivation, and the magnitudes of the rate constants for inactivation relative to the experimentally determined value for the reaction of diethylpyrocarbonate with N alpha-acetylhistidine (2.2 M-1 s-1), all suggested that enzyme inactivation occurs solely by modification of histidine residues. Comparison of the experimental plot of residual fractional activity versus the number of modified histidine residues per subunit with simulated plots for three hypothetical models, each predicting biphasic kinetics, indicated that inactivation results from the modification of at most one essential histidine residue per subunit, although it appears that other (non-essential) histidines react independently. This histidine is thought to be His-242 and is present in the active site. Evidence in support of its role in catalysis is briefly discussed. Both 6-phosphogluconate and organic phosphate protect against inactivation, and a kinetic analysis of the protection indicated a dissociation constant of 2.1 X 10(-6) M for the enzyme--6-phosphogluconate complex. NADP+ also protected, but this might be due, at least in part, to a reduction in the effective concentration of diethylpyrocarbonate.     

Chemical and kinetic evidence for an essential histidine in horseradish peroxidase for iodide oxidation.

Horseradish peroxidase (HRP), when incubated with diethylpyrocarbonate (DEPC), shows a time-dependent loss of iodide oxidation activity. The inactivation follows pseudo-first order kinetics with a second order rate constant of 0.43 min-1 M-1 at 30 degrees C and is reversed by neutralized hydroxylamine. The difference absorption spectrum of the modified versus native enzyme shows a peak at 244 nm, characteristic of N-carbethoxyhistidine, which is diminished by treatment with hydroxylamine. Correlation between the stoichiometry of histidine modification and the extent of inactivation indicates that out of 2 histidine residues modified, one is responsible for inactivation. A plot of the log of the reciprocal half-time of inactivation against log DEPC concentration further suggests that only 1 histidine is involved in catalysis. The rate of inactivation shows a pH dependence with an inflection point at 6.2, indicating histidine derivatization by DEPC. Inactivation due to modification of tyrosine, lysine, or cysteine has been excluded. CD studies reveal no significant change in the protein or heme conformation following DEPC modification. We suggest that a unique histidine residue is required for maximal catalytic activity of HRP for iodide oxidation.     

The presence of histidine residues at or near the C1q binding site of rabbit immunoglobulin G

Treatment of covalently crosslinked rabbit IgG oligomers with diethylpyrocarbonate resulted in the loss of their C1q binding activity. The inactivation was a first-order process with respect to time in the range 0-8 min, and modifier concentration from 0 to 2.39 mM. Hydroxylamine treatment of diethylpyrocarbonate-treated IgG oligomers led to 80% recovery of their C1q binding activity. Diethylpyrocarbonate treatment of IgG oligomers had little effect on their absorbance at 278 nm, but led to an increase in their absorbance at 242 nm. The apparent pKa of the modified residues was 6.91 +/- 0.12. These data are consistent with diethylpyrocarbonate modification of histidine residues leading to loss of C1q binding activity in rabbit IgG oligomers. Modification of four histidine residues per IgG molecule was associated with the loss of C1q binding activity. Thus, there may be two histidine residues at or near the C1q binding sites in the CH2 domains of rabbit IgG.     

Role of arginine and histidine residues in the biological activity of botulinic neurotoxin A

Treatment of botulinic neurotoxin A with cyclohexanedione demonstrated that modification of 5 to 10 arginine residues does not change the neurotoxin toxicity, while after modification of 15-20 arginine residues the toxicity is decreased by 40-50% of the original value. Butanedione exerts a stronger detoxicating effect on neurotoxin than cyclohexanedione. The molecular conformation of the modified toxin derivatives and their precipitability upon interaction with antisera against toxin and toxin fragments does not change thereby. The non-toxic derivatives of toxin containing 40 modified arginine residues possess a partial serological affinity for the original toxin in a reaction with antiserum against toxin but do not interact with the antifragment sera. The molecular conformation of these preparations is changed considerably. It is assumed that one or two arginine residues are located near the toxic site of the neurotoxin molecule and are also components of its antigenic determinants. Modification of histidine residues in the neurotoxin molecule by diethylpyrocarbonate is accompanied by a decrease of its toxicity. An additional 10% toxicity is revealed upon modification of 11-13 histidine residues. The molecular conformation of the modified derivatives of neurotoxin and their precipitability do not change thereby. It is probable that 1 or 2 histidine residues are located at or near the toxic site. The data obtained suggest that histidine residues are not localized in antigenic determinants of the neurotoxin molecule.     

An essential histidine residue for the activity of UDPglucose 4-epimerase from Kluyveromyces fragilis.

UDPglucose 4-epimerase from Kluyveromyces fragilis was completely inactivated by diethylpyrocarbonate following pseudo-first order reaction kinetics. The pH profile of diethylpyrocarbonate inhibition and reversal of inhibition by hydroxylamine suggested specific modification of histidyl residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of 1 essential histidine residue to be responsible for loss in catalytic activity of yeast epimerase. No major structural change in the quarternary structure was observed in the modified enzyme as shown by the identical elution pattern on a calibrated Sephacryl 200 column and association of coenzyme NAD to the apoenzyme. Failure of the substrates to afford any protection against diethylpyrocarbonate inactivation indicated the absence of the essential histidyl residue at the substrate binding region of the active site. Unlike the case of native enzyme, sodium borohydride failed to reduce the pyridine moiety of the coenzyme in the diethylpyrocarbonate-modified enzyme. This indicated the presence of the essential histidyl residue in close proximity to the coenzyme binding region of the active site. The abolition of energy transfer phenomenon between the tryptophan and coenzyme fluorophore on complete inactivation by diethylpyrocarbonate without any loss of protein or coenzyme fluorescence are also added evidences in this direction.     

Modification of pig liver dimeric dihydrodiol dehydrogenase with diethylpyrocarbonate and by rose bengal-sensitized photooxidation: evidence for an active-site histidine residue.

Dihydrodiol dehydrogenase from pig liver was inactivated by diethylpyrocarbonate (DEP) and by rose bengal-sensitized photooxidation. The DEP inactivation was reversed by hydroxylamine and the absorption spectrum of the inactivated enzyme indicated that both histidine and tyrosine residues were carbethoxylated. The rates of inactivation by DEP and by photooxidation were dependent on pH, showing the involvement of a group with a pKa of 6.4. The kinetics of inactivation and spectrophotometric quantification of the modified residues suggested that complete inactivation was caused by modification of one histidine residue per active site. The inactivation by the two modifications was partially prevented by either NADP(H) or the combination of NADP+ and substrate, and completely prevented in the presence of both NADP+ and a competitive inhibitor which binds to the enzyme-NADP+ binary complex. The DEP-modified enzyme caused the same blue shift and enhancement of NADPH fluorescence as did the native enzyme, suggesting that the modified histidine is not in the coenzyme-binding site of the enzyme. The results suggest the presence of essential histidine residues in the catalytic region of the active site of pig liver dihydrodiol dehydrogenase.     

Chemical modification of histidine, tyrosine, tryptophan and cysteine residues in carp (Cyprinus carpio) muscle enolase

Enolase from carp (Cyprinus Carpio) muscle was modified by diethylpyrocarbonate, tetranitromethane, N-bromosuccinimide and 5,5'-dithiobis(2-nitrobenzoic acid). The extent and rate of modification and its effect on the enzyme activity were determined. Modification of histidine, tyrosine and tryptophan residues caused complete inactivation of the enzyme; Mg2+ as well as 2-phosphoglycerate markedly altered the rates of modification and inactivation. The above-mentioned amino acid residues seem to be essential for the functioning of muscle enolases. Modification of cysteine residues had no effect on the enolase activity.     

Chemical modification of dopamine beta-hydroxylase

Dopamine beta-hydroxylase (3,4- dihydroxyphenylethylamine ,ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) is the terminal enzyme in the biosynthetic pathway of norepinephrine. Chemical modification studies of this enzyme were executed to investigate contributions of specific amino-acid side-chains to catalytic activity. Sulfhydryl reagents were precluded, since no free cysteine residue was detected upon titration of the denatured or native protein with 2-chloromercuri-4-nitrophenol. Incubation of enzyme with diazonium tetrazole caused inactivation of the protein coupled with extensive reaction of lysine and tyrosine residues. Reaction with iodoacetamide resulted in complete loss of enzymatic activity with reaction of approximately three histidine residues; methionine reaction was also observed. Modification of the enzyme using diethylpyrocarbonate resulted in complete inactivation of the enzyme, and analysis of the reacted protein indicated a loss of approx. 1.7 histidine residues per protein monomer with no tyrosine or lysine modification observed. The correlation of activity loss with histidine modification supports the view that this residue participates in the catalytic function of dopamine beta-hydroxylase.     

Chemical modification of essential histidine residues in aspartase with diethylpyrocarbonate

Aspartase purified from Escherichia coli W cells was inactivated by diethylpyrocarbonate following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with NH2OH, the enzyme activity was completely restored. The difference absorption spectrum of the modified vs. native enzyme preparations exhibited a prominent peak around 240 nm. The pH-dependence of the inactivation rate suggested that an amino acid residue having a pK value of 6.6 was involved in the inactivation. These results indicate that the inactivation was due to the modification of histidine residues. L-Aspartate and fumarate, substrates for the enzyme, and the Cl- ion, an inhibitor, protected the enzyme against the inactivation. Inspection of the spectral change at 240 nm associated with the inactivation in the presence and absence of the Cl- ion revealed that the number of histidine residues essential for the enzyme activity was less than two. Partial inactivation did not result in an appreciable change in the substrate saturation profiles. These results suggest that one or two histidine residues are located at the active site of aspartase and participate in an essential step in the catalytic reaction.     

Essential histidine residue in 3-ketosteroid-delta 1-dehydrogenase.

The variation with pH of kinetic parameters was examined for 3-ketosteroid-delta 1-dehydrogenase from Nocardia corallina. The Vmax/Km profile for 4-androstenedione indicates that activity is lost upon protonation of a cationic acid-type group with a pK value of 7.7. The enzyme was inactivated by diethylpyrocarbonate at pH 7.4 and the inactivation was substantially prevented by androstadienedione. Analyses of reactivation with neutral hydroxylamine, pH variation, and spectral changes of the inactivated enzyme revealed that the inactivation arises from modification of a histidine residue. Studies with [14C]diethylpyrocarbonate provided support for the idea that the 1-2 essential histidine residues are essential for the catalytic activity of the enzyme. Dye-sensitized photooxidation led to 50% inactivation of the enzyme with the decomposition of two histidine residues. This inactivation was also prevented by androstadienedione. Dancyl chloride caused a loss of the enzyme activity. Modifiers of glutamic acid, aspartic acid, cysteine, and lysine did not affect the enzyme activity. Butanedione and phenylglyoxal in the presence of borate rapidly inactivated the enzyme, indicating that arginine residues also have a crucial function in the active site. The data described support the previously proposed mechanism of beta-oxidation of 3-ketosteroid.     

Inactivation of Acinetobacter calcoaceticus acetate kinase by diethylpyrocarbonate

Acetate kinase purified from Acinetobacter calcoaceticus was inhibited by diethylpyrocarbonate with a second-order rate constant of 620 M-1.min-1 at pH 7.4 at 30 degrees C and showed a concomitant increase in absorbance at 240 nm due to the formation of N-carbethoxyhistidyl derivative. Activity could be restored by hydroxylamine and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.64. Complete inactivation of acetate kinase required the modification of seven residues per molecule of enzyme. Statistical analysis showed that among the seven modifiable residues, only one is essential for activity. 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuryphenylsulfonate, N-ethylmaleimide and phenylglyoxal did not affect the enzyme activity. These results suggest that the inactivation is due to the modification of one histidine residue. The substrates, acetate and ATP, protected the enzyme against inactivation, indicating that the modified histidine residue is located at or near the active site.     

Studies of histidine residues in soybean (Glycine max) urease

Soybean urease has been investigated extensively to reveal the presence of histidine residue (s) in the active site and their potential role in the catalysis. The spectrophotometric studies using diethylpyrocarbonate (DEP) showed the modification of 11.76 ± 0.1 histidine residues per mole of native urease. Therefore, the results are indicative of the presence of twelve histidine residues per urease molecule. It is presumed that the soybean urease, being a hexameric protein possess two histidine residues per subunit. Correlation plot showed that the complete inactivation of soybean urease corresponds to the modification of 1.97 histidine residues per subunit. Further, double logarithmic plot of kapp versus DEP concentration has resulted in a linear correlation and thereby demonstrating that only one of the two histidine residues per subunit is catalytically essential. Significant protection has been observed against inactivation when urea or acetohydroxamate (AHA) is incubated with DEP treated urease. The studies have demonstrated the presence of one histidine residue at the active site of soybean urease and its significance in catalysis.