Studies on chemical modifications of enzymes with O-Benzoquinone. A new reaction of the Quinone with Methionine

Abstract

The thesis deals mainly with studies on a new reaction of o-benzoquinone with methionine. o-Benzoquinone is a highly reactive compound which takes part in many complex reactions with proteins. Some of the functional groups that are known to be involved in these reactions are the amino, imino and sulfhydryl groups. Apart from these, in the present investigation, a new reaction of o-benzoquinone with the thioether group of methionine was detected. A detailed study of this reaction has now been made with a view to elucidate the structure of the reaction product, determine the kinetics of the reaction and utilize the reaction for chemical modification of methionine residues of enzymes with particular reference to ribonuclease A (RNase A). This reaction has also been made use of for detecting differences in the reactivity of methionine residues of structurally similar proteins such as RNase A and RNase S. In this investigation crystalline o-benzoquinone (oBQ) prepared by the oxidation of catechol has been used. Freshly prepared quinone which was obtained as bright red crystals was found to be stable in a suspension of anhydrous ether at 0°C for more than four hours. The quinone was found to have characteristic absorption spectra in solvents such as ether, chloroform, hexane and glacial acetic acid with absorption maxima in the region of 380 m?. As could be judged from the constancy of the absorption spectra with time the quinone was found to be relatively stable at room temperature in these solvents. In glacial acetic acid, the quinone had the absorption maxima at 380 m? and the molar extinction value of the quinone at 380 m? was determined to be 1800. The quinone was less stable in aqueous solvents as well as in solvents like alcohol and formic acid. In aqueous solutions at pH below 2 the quinone was stable for periods up to 10 minutes. Above this pH the rate of decomposition increased progressively with pH. In view of this, the present studies on the reaction of the quinone with amino acids and proteins have generally been carried out either in glacial or aqueous acetic acid or in dilute aqueous hydrochloric acid solutions. Preliminary studies showed that oBQ reacted with enzymes RNase and lysozyme at acidic pH to give products the spectra of which indicated introduction of phenolic chromophores into the protein. The spectra characteristics of the oBQ-treated RNase were found to be unaffected either by performic acid oxidation or by reduction and carboxymethylation. A chemical reaction between oBQ and the protein was therefore clearly apparent. The reaction of oBQ with amino acids in 20% acetic acid solutions has been studied. When the amino acids were reacted with oBQ and subsequently treated with a reducing agent, the amino acids that were found to be modified were methionine and cysteine. The reaction products of these two amino acids were found to have distinct chromatographic mobility and characteristic absorption spectra. It was evident from these studies that the reaction of oBQ involved the thioether and the sulfhydryl groups of these amino acids and the incorporation of aromatic moieties to the amino acids. N-Acetylmethionine (NAM), N-acetylmethionine methyl ester (NAME) and methionine containing dipeptide N-carbobenzoxy methionyl glycine ethyl ester were also found to give condensation products with oBQ. oBQ derivatives of NAM, NAME, cysteine and thiourea have been prepared and were found to be chromatographically homogeneous. The reaction of oBQ with cysteine was found to be associated with side reactions. The oBQ derivative of cysteine, namely S-(3,4-dihydroxyphenyl)cysteine hydrochloride and oBQ derivative of thiourea, namely S-(3,4-dihydroxyphenyl)isothiourea acetate have been obtained in crystalline form. A detailed study has been made of the electrophoretic, spectral and potentiometric titration characteristics of the oBQ derivative of NAM. It was found that the oBQ derivative of NAM prepared in presence of HCl contained one equivalent of chloride per mole of the derivative. The derivative was also found to have an excess positive charge at pH 2 as compared to NAM. This was clearly indicative of the presence of a sulfonium group in this derivative. The acid hydrolysate of the derivative was found to yield homoserine and homocysteine further confirming the sulfonium grouping in the derivative. In acidic solutions the oBQ derivative of NAM was found to have an absorption spectrum with ? at 250 m? and 287 m?, the molar extinction coefficient being 7.57 × 10³ and 5.45 × 10³, respectively. At pHs above 6.0 spectral changes characteristic of phenolic ionisation were observed. These changes were found to be of two categories: (i) a reversible ionisation of a phenolic group with pK of 6.95 and (ii) an irreversible ionisation above pH 9.0. Potentiometric titration data of the oBQ derivative of NAM showed that the derivative has three ionisable groups with pKs of 3.0, 6.9 and 11.25 which could be assigned to a carboxyl, a phenolic hydroxyl with a low pK, and another phenolic hydroxyl, respectively. On the basis of the above results, the following structure has been proposed for the oBQ derivative of NAM: HO CH? S-CH?-CH?-CH-NH-COCH? or S-(3,4-dihydroxyphenyl)-N-acetylmethionine sulfonium chloride. The elementary analysis of the oBQ derivative of NAM was found to conform to the above structure. Kinetic studies on the reaction between oBQ and NAM have been carried out. In glacial acetic acid the reaction was found to be second order with a rate constant of 50 M?¹min?¹ at 21°C. In aqueous acetic acid as well as in aqueous solutions at pH 1.0 the reaction was found to be extremely fast. Reaction of oBQ with enzymes RNase, lysozyme and ?-chymotrypsin was found to affect their enzymatic activity. The inactivation of the enzymes was dependent to a very great extent on the pH of the reaction medium. Enzymes RNase and ?-chymotrypsin were inactivated at pHs below 3.0 and at pH 7.0 but not at pH 4.0. Lysozyme was found to be inactivated only in the region of 7.0 and not at lower pHs. The pH-dependence of inactivation was indicative of the differences in the nature of the reaction at acidic and at neutral pH regions. Spectral studies on the RNase derivative obtained by oBQ reaction at acid pH were found to indicate the reaction of the methionine residues of the protein. oBQ derivatives of RNase A have been prepared by carrying out the reaction at pH 1.0. Amino acid analysis of these derivatives showed that the only amino acid residue that had undergone chemical modification as a result of oBQ reaction was methionine. The extent of methionine modification in these derivatives determined either by amino acid analysis or by difference spectral measurements was found to be the same. At pH 1.0 and under controlled conditions of oBQ concentration and reaction time an oBQ-reaction product of RNase A with an average modification of 1.8 methionine residues per mole hasbeen prepared. This product did not contain any unreacted RNase A. By chromatographic fractionation a product having one methionine residue modified per mole of the protein has been isolated. Analysis of the tryptic peptides of this product indicated modification of only methionine residues. This analysis also showed that this product contained monosubstituted (methionine) derivatives of RNase A, the modification being distributed among the methionine residues at different positions in the protein chain. Thus the reaction of oBQ at pH 1.0 was found to be nearly equally favoured at all the four methionine residues of RNase A. Methionine residues of RNase A as well as those of RNase S were found to react easily with the quinone at pH 1.0. However, at pH 3.0, even at high concentrations of the quinone, the methionine residues of RNase A were not modified. In contrast, the methionine residues of RNase S, S-protein and S-peptide were found to react at this pH. Apparently the methionine residues of RNase A are present in an environment (hydrophobic) which does not favour reaction with the quinone. The reactivity of the methionine residues of RNase S at pH 3.0 could be attributed either to the dissociation of RNase S to S-protein and S-peptide (both of which have been found to be reactive to the quinone) or to the presence of one or more methionine residues of undissociated RNase S in an environment which favours reaction with the quinone. In conclusion, the present investigation shows that one of the reactions that o-benzoquinone is capable of taking part with proteins involves the thioether group of methionine residues. This reaction proceeds at an extremely fast rate at acidic pHs leading to the incorporation of the aromatic moiety at the sulfur atom of the methionine and formation of a sulfonium derivative. In reactions of oBQ with methionine-containing proteins, the structure of the protein and the environment in which the methionine residues are placed have been observed to have an influence on the reactivity of these residues to the quinone.