Reactivity of Glycyl-Amino Acids Toward Hydroxyl Radical. in Neutral Aqueous Solutions. T. MASUDA, N. IWASHITA, H. SHINOHARA and M.

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1 Reactivity of Glycyl-Amino Acids Toward Hydroxyl Radical in Neutral Aqueous Solutions T. MASUDA, N. IWASHITA, H. SHINOHARA and M. KONDO Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Setagaya, Tokyo 1-08, Japan (Received June 15, 1977; Revised version received July 29, 1977) Reactivity of dipeptide/hydroxyl radicals/rate constant Rate constants for reactions of hydroxyl radicals with several glycyl-amino acids were determined by a competition method using p-nitrosodimethylailine as a reference compound. For glycyl-aliphatic amino acids, the enhancement of reactivity was observed as compared with the corresponding free amino acids. The reactivity was explained qualitatively in terms of partial reactivities assigned to each C-H bond of the dipeptides. For glycyl-aromatic amino acids, the rate constants were found to be almost equal to those of the corresponding free amino acids. The reactivity of a protein toward hydroxyl radical was well understood by summation of the rate constants, corrected by steric factors, of amino acid residues located on surface of the protein. The enhanced reactivity of the aliphatic peptides was interpreted in terms of the difference in interaction energy between NH2- and NH3+-forms of an aliphatic amino acid, which was calculated for the system including glycine and hydroxyl radical ac cording to CNDO/2 method. INTRODUCTION In previous papers,'," we reported that a rate constant for reaction of a protein with a hydroxyl radical could be estimated from the summation of rate constants, corrected by steric factors, of amino acid residues which were found on surface of the protein molecule. The estimation was based on the assumption that the rate con stants determined for free amino acids could be used for the calculation on behalf of those for amino acid residues present in peptide and steric factors could be evaluated from cubic model. The reactivity of an aliphatic amino acid residue in peptide, how ever, was found to be higher than that of the corresponding free amino acid." The enhancement of reactivity was explained mainly in terms of disappearance of the retarding effect owing to a protonated amino group. Therefore, the summation of the rate constants, corrected by steric factors, of amino acid residues present in peptide should be carried out to obtain an accurate estimate of the reactivity of pro tein toward hydroxyl radical. For simple glycyl-aliphatic amino acids, the rate con stants were already determined and reported in the previous paper." In the present investigation, determination of the rate constants for reactions of

2 hydroxyl radicals with glycyl-aromatic amino acids and glycyl-aliphatic amino acids having functional group(s) other than a-carboxyl and a-amino was carried out in neutral aqueous solutions and the validity of the estimation based on the summation of rate constants of amino acid residues located on surface of protein was examined, when the rate constants determined for amino acid residues present in peptide were used for the calculation. Furtheremore, a quantum-chemical interpretation of the re tarding effect of a protonated amino group on the reaction rate of hydrogen atom abstraction by hydroxyl radical was attempted by comparing the interaction energy of the system including a glycine and a hydroxyl radical. MATERIALS AND METHOD Glycyl-amino acids were obtained from Nutritional Biochemicals Corp. and used without further purification. p-nitrosodimethylaniline (PDNA) was obtained from the British Drug Houses Ltd. and recrystallized twice from ether. Solutions for irradiation were prepared with triply distilled water and adjusted to ph 6-7 by adding 0.1 N NaOH or 0.1 N HZSO4. Irradiation condition was almost the same as described in the previous papers.'-') The rate constants for reactions of hydroxyl radicals with the dipeptides were determined according to the method of Kraljic and Trumbore,4' taking k(oh+pnda) =1.25x 1010 M-1 sec-1.5, Calculation of the interaction energy was carried out with FACOM S com puter according to the CNDO/2 method." RESULTS AND DISCUSSION Rate constants for reactions of hydroxyl radicals with several glycyl-amino acids were determined in aqueous solutions by the competition method using p-nitrosodime thylaniline as a reference substance and are shown in Table 1. For some of the peptide listed in Table 1, the rate constants determined at ph 2 were already given by Scholes et al.") The agreement of the values determined at ph 2 with the present ones is fairly well, except of glycylphenylalanine and glycylmethionine. Since the rate constants for reactions of hydroxyl radical with phenylalanine and methionine were reported by the same authors") to be 7.0X 103 and 5.9x 109 M-1 sec-1 respectively, 8.2 x10' and 4.0X 109 M-1 sec-1 seem to be too small for the dipeptides. As shown in Table 1, the rate constants of the glycyl-aliphatic amino acids are generally larger than those of the corresponding amino acids except glycylthreonine, even if the partial rate constant of glycyl moiety (4X 10` M-1 sec-1)3' is substracted from those for the glycyl-amino acids. On the other hand, the rate constants of glycyl-aromatic amino acids and glycyl methionine are almost equal to or slightly smaller than those of the corresponding amino acids. For glycyltyrosine, the present value is somewhat uncertain, because poor solubility of the peptide at neutral ph leads to uncertainity in concentration. In the previous works,',") the reactivity of an aliphatic amino acid or peptide was

3 Table 1 The rate constants for reactions of hydroxyl radicals with glycyl-amino acids deter mined in neutral squeous solutions and the estimated rate constants from the partial rate constants assigned to each C-H bond of aliphatic amino acids and peptides a) The values were estimated from the partial rate constants assigned to each C-H bond of glycyl-amino acids and amino acids as reported in the previous paper." b) Reference (10). The originally reported values are corrected for the recent value of koh+thymine (4.7 x 109 M-1 sec-1). c) The rate constants of the C-terminal amino acids of the corresponding dipeptides. The values were determined at ph and reported in the previous paper.') explained in terms of partial reactivities assigned to each C-H bond of the amino acid or peptide molecule. And the enhancement of reactivity of an amino acid residue present in peptide was explained by disappearance of the retarding effect due to the protonated amino group. When the same argument is applied to the present results, the estimated values indicated in the fourth column of Table 1 are obtained. They were calculated by the following equation. kpeptide-k'lycy+ ki fi i where kg,ycy, represents the partial rate constant for glycyl moiety and k; for ith C-H bond of C-terminal amino acid residue. k; were evaluated from the rate constants of simple glycyl-aliphatic amino acids." fi is a correction factor due to the effect of functional group(s) other than a-carboxyl and a-amino. As compared with the case of simple glycyl-aliphatic amino acids," the agreement of the estimated values with the observed is not good, especially for glycylthreonine and glycylserine. For other glycyl-amino acids, the disagreement is not significant. Since the hydroxyl group of seryl or threonyl residue is known to form an intramolecular hydrogen bond with peptide bond in myoglobin,12) the hydrogen bond formed may restrict the reaction with hydroxyl radical. The rate constants for reactions of hydroxyl radicals with glycyl-aromatic amino

4 acids are almost equal to those of the corresponding amino acids. Since hydroxyl radicals undergo the addition reaction with aromatic compounds, the result suggests that ;z-electron state of an aromatic ring is hardly affected by the protonated amino group of aliphatic moiety of the molecule. Unfortunately glycyl-amino acids of glutamine, arginine, lysine, and cystine could not be obtained commercially. The rate constants of the corresponding amino acids were, therefore, used as the approximate values for those of the residues in the cal culation of the rate constant of protein. As shown in Table 2, the present estimated values are a little larger than those calculated from the rate constants of amino acids. Furthermore, if the rate constants of the unavailable peptides are used for the cal culation, the estimated values will be larger than those shown in Table 2, because of the expected enhancement of the reactivity. This discrepancy seems to be caused by overestimation of steric factors based on a simple cubic model." The cubic model is not adequate especially for the evaluation of steric factors of long molecules such as leucine, isoleucine, lysine, or arginine. When a hydroxyl radical attacks such a residue along the extended line of molecular axis, the steric hindrance may depend on the position of C-H bond. The more inside the C-H bond stands, the greater the steric hindrance. Therefore, to obtain the steric factor accurately, a solid angle ad mitted for the radical attack should be evaluated for each C-H bond of the residue on surface of protein and compared with that for the corresponding amino acid. Although the calculation of such solid angle is theoretically possible with computer and X-ray crystallographic data of protein, it was not achieved in the present work because of necessity of a huge computer program. However, the calculation is rea sonably expected to give smaller steric factors than those obtained from the cubic model, in which the equal probability is given to the direction of molecular axis as well as the directions perpendicular to the axis. The enhancement of the reactivity of amino acid residue in peptide was interpreted Table 2 The estimated rate constants for reactions of hydroxyl radicals with enzyme proteins a) The values were estimated from the rate constants of amino acid residues in peptides b) The values were estimated from the rate constants of amino acids. These values were reported in the previous paper.') c> The values were determined experimentally and the references from which the values were cited are seen in the previous paper.')

5 by disappearance of the retarding effect due to a protonated amino group," because for aliphatic amino acids larger rate constants were generally reported in alkaline solution than in neutral solution. For example, the rate constant for reaction of hydroxyl radical with glycine was determined by pulse radiolysis using thiocyanate ion to be (1.7±0.17) X 10' M-1 sec-1 at ph 5.8 and (1.8±0.15) X 109 M-1 sec-1 at ph ' Since pk2 of glycine is 9.60 and pka of hydroxyl radical is 11.9, the rate constant determined at ph 9.5 approximately corresponds to the reaction of hydroxyl radical with the equimolar mixture of NH,' and NH,-forms of glycine. This suggests that the enhancement of reactivity toward hydroxyl radical is caused by deprotonation of amino group. Wigger et a1.13' reported that CH,-NH, reacts rapidly with hydroxyl radical to form CH3-NH radical but CH,-NH,' reacts slowly with the radical. In the case of NH,-form of amino acid, it should be considered that hydroxyl radical may attack the amino group to give N-radical. However, ESR studies confirmed the C radicals in alkaline solution of aliphatic amino acids.","' The results indicate that the attack of hydroxyl radical on amino group can be neglected in discussion of hydrogen abstraction reaction. The partial reactivity which was found in the reaction of aliphatic amino acid with hydroxyl radical could be well explained by the interaction energy between an aliphatic amino acid and a hydroxyl radical at a close contact dis tance."' A similar calculation was carried out to interpret the effect of a protonated amino group on the reactivity toward hydroxyl radical. The geometries used for the calculation are shown in Fig. 1. N, C, C, 0 and 0 atoms in NH,'-form or H, H, N, C, C, 0, and 0 atoms in NH,-form lie on a plane. In the system including a glycine and a hydroxyl radical, the distance of C-H10 (C-H9) and O-H10 (0-H9) shown in Fig. 1 were already confirmed to give the minimum energy."' The geometry may be roughly approximated as an activated state. The bond angle of H-0-H10 (HOH9) was also con firmed to give the minimum energy in the previous work."' The total energy shown in Table 3 was obtained as a sum of one and two-atom terms,'''' and two-atom terms (interatomic interaction energies) are separately shown to indicate the contribution of the terms on the reactivity. The difference in interaction energy between NH3+ and NH2-glycine, calculated from the total energies, is about 8.1 kcal mol-1 and that from the two-atom terms is about 8.7 kcal mol-1. The reactivity of the equimolar mixture of NH3+ and NH2-glycine is about a hundred times greater than that of NH,'-glycine and hence the difference in activation energy at 300 K was evaluated to be approxim ately 3 kcal mol-1 if assumed that the pre-exponential terms are identical in Arrhenius equation. The difference in interaction energy obtained theoretically is considerably larger than that estimated from the experimental result. This may be attributable to the insufficient approximation of the configuration of activated complex. However, the argument based on the interaction energy appears to be relevant to the qualita tive explanation of the retarding effect of protonated amino group. Two-atom terms gave almost the same difference in energy as that from total energy. The difference in stabilization energy due to H-0 bond formation, Ex-o, is rather small between NH3+ and NH2-glycine, while the energy change due to stretching of C-H bond, Ec-x, differs

6 Fig. 1. Geometries of NH3+-glycine, NH2-glycine, and their reaction complexes with hydroxyl radical, used for the calculation of interaction energies. a) NH3+-glycine. b) NH3+-glycine and hydroxyl radical complex. c) NH2-glycine. d) NH2-glycine and hydroxyl radical complex. Figures inserted into the geometries indicate the interatomic distance in A and bond angles, respectively. Table 3 The interaction energies calculated by CNDO/2 method for the system of glycine and hydroxyl radical a) The interaction energy calculated from the total energies according to the following relation. E=Egly+ox (Egiy+Eox) Where Eox is and Egly are and for NH3+ and NH2-glycine, respectively. Egly+ox are and for NH3+ and NH2-glycine, respectively. b) Two-atom terms for Hlo (H9) atom and a-carbon atom of glycine. c) Two-atom terms for H10 (H9) atom of glycine and oxygen atom of hydroxyl radical.

7 0 2.50A from each other. Therefore, it is concluded that the reactivity toward hydroxyl radical depends mainly on the energy needed for stretching of C-H bond of glycine. Since the reactivity of glycine toward hydroxyl radical was determined in aqueous solution, the effect of neighboring water molecules on the reactivity should be con sidered. For the purpose, we calculated the interaction energies between glycine and hydroxyl radical when a water molecule approaches to the hydrogen atom of amino group along the extended axis of H-N, using the geometries reported by Imamura et al. 1 7' for the calculation of stabilization energy of glycine-water system. The inter action energy was calculated at 2.60 A of the distance O-H-N for NH,'-glycine and at for NH2-glycine respectively, because the minimum energy was obtained at the distances, respectively. The difference in interaction energy was 11.3 kcal mol-1 from the total energies and 8.7 kcal mol-' from the two-atom terms. Since the retarding effect due to carboxyl group is rather small,',"' it may be concluded that the reactivity is not significantly affected by the neighboring water molecules. Table 4 The interaction energies calculated by CNDO/2 method for the system of glycine, hydroxyl radical, and water a) From the total energies. Egly are and for NH3+ and NH2 glycine, respectively. Egly+ox are and for NH3+ and NH2 glycine, respectively. b) Two-atom terms for H10 (H9) atom and a -carbon atom of glycine-water. 0> Two-atom terms for H10 (H9) atom of glycine-water and oxygen atom of hy droxyl radical. REFERENCES 1. T. Masuda, S. Nakano and M. Kondo (1973) Rate constants for the reaction of OH radicals with the enzyme proteins as determined by the p-nitrosodimethylaniline method. J. Radiat. Res., 14: T. Masuda, S. Nakano and M. Kondo (1974) Reactivities of enzymes toward hydroxyl radicals. J. Radiat. Res., 15: T. Masuda, K. Yoshihara, H. Shinohara and M. Kondo (1976) Reactivity of aliphatic peptides toward hydroxyl radicals in aqueous solution. J. Radiat. Res., 17: I. Kral jic and C. N. Trumbore (1965) p-nitrosodimethylaniline as an OH radical scavenger in radiation chemistry. J. Am. Chem. Soc., 87: R. L. Willson, C. L. Greenstock, G. E. Adams, R. Wageman and L. M. Dorfman (1971) The standardization of hydroxyl radicals rate data from radiation chemistry. Int. J. Radiat. Phys. Chem., 3 :

8 6. J.A. Pople and R.K. Nesbet (1954) Self-consistent orbitals for radicals. J. Chem. Phys., 22: J.A. Pople, D.P. Santry and G.A. Segal (1965) Approximate self-consistent molecular or bital theory. I. Invariant procedures. J. Chem. Phys., 43: S129-S J. A. Pople and G.A. Segal (1965) Approximate self-consistent molecular orbital theory. II. Calculations with complete neglect of differential overlap. J. Chem. Phys., 43: S136-S J.A. Pople and G.A. Segal (1966) Approximate self-consistent molecular orbital theory. III. CNDO results for AB2 and AB, systems. J. Chem., Phys., 44: G. Scholes, P. Shaw, R. L. Willson and M. Ebert (1965) Pulse radiolysis studies of aqueous solution of nucleic acid and related substances. Pulse Radiolysis (ed. by M. Ebert, J. P. Keene, A. J. Swallow and J. H. Baxendale) Academic Press, London and New York. pp T. Masuda, S. Nakano, K. Yoshihara and M. Kondo (1976) Partial reactivities observed in the hydrogen abstraction reaction of hydroxyl radicals from aliphatic amino acids. J. Radiat. Res., 17: J. C. Kendrew (1962) Side-chain interactions in myoglobin. Brookhaven Symp. Biol., 15: A. Wigger, W. Gri nbein, A. Henglein and E. J. Land (1969) Pulseradiolytische Untersuch ung primarer Schritte der Oxidation von Aminen in wassriger Losung. Z. Naturforschg., 24b: H. Paul and H. Fischer -(1969) Elektronenspinresonanz kurzlebiger Radikale aus einigen Aminosauren and Amiden. Ber. Bunsenges. Phys. Chem., 73: R. Poupko, A. Loewenstein and B.L. Silver (1971) Electron spin resonance study of radi cals derived from simple amines and amino acids. J. Am. Chem. Soc., 93: H. Shinohara, A. Imamura, T. Masuda and M. Kondo (to be published). 17. A. Imamura, H. Fujita and C. Nagata (1969) The electronic structures of glycine in the isolated state and in water. Bull. Chem. Soc. Japan, 42: