Kinetics of the Reaction of Nitrous Acid with Model Compounds and Proteins, and the Conformational State of N-terminal Groups in the Chymotrypsin Family

Abstract

of the reaction of nitrous acid with model compounds and proteins, and the conformational state of N-terminal groups in the chymotrypsin family. Can. J. Biochem. 50,1282Biochem. 50, -1296.The kinetics of the reaction of nitrous acid at 4 O and pH 4.8 with various amino acids, peptides, and proteins were studied. The reaction with isoleucine methyl ester was found to have a linear dependence on the square of the H O N 8 concentration showing that N28, was the reactive species. Third order nitrosation rate constants of primary amino groups showed a correlation with their pKvalues. They were calculated for the concentration of the unprotonated species to give intrinsic reactivities. The rate sf nitrosation of acetyltryptophan to give N-nitrosoacetyltryptophan was found to be a linear function of the nitrous acid concentration. This nitrosation therefore follows a different mechanism. The reaction of nitrous acid with tyrosine residues was examined by sptrophotometry. The reaction was negligible compared to that of other groups. Acetylhistidine and imidazole did not react. Reactivities for a-amino groups, &-amino groups, and other residues in proteins were compared. The confornational state of the N-terminal residues in serine proteinases, as revealed from theit reactivities, is discussed in detail. It is concluded that nitrous acid reacts preferentially with "surface" residues and is a useful tool for exploring conformational states of reactive groups in proteins, especially a-amino groups and indole rings.A., et HOFMANN, T. Kinetics of the reaction of nitrous acid with model compounds and proteins, and the conformational state of N-terminal groups in the chymotrypsin family. Can. J. Biochem. 50,1282Biochem. 50, -1296Biochem. 50, (1972.Nous avons ttudik le cinetique de la rtaction de l'acide nitreux a 4" et a pH 4.8 avec divers acides amints, des peptides et des protkines. La reaction avec I'ester mtthylk de l'isoleucine montre une dkpenbnce lintaire avec le c a r t de la concentration de HONO. La substance qui rkagit est donc le N203. Les constantes de vitesse de nitrosation de troisitme ordre des groupements amints primaires sont en relation avec leurs valeurs de pK. NOW les avons calculkes pour la concentration des composts non protonises pour donner les rkactivites intrinkques. La v i t e s~ de nitrosation de l'acttyltryptophanne en N-nitrosoacttyltryptophanne est fonction lintaire de la concentration de B'acide nitreux. Cette nitrosation suit donc un mkcanisme diffkrent. La rkaction de l'acide nitreux avec les rCsidus de tyrosine est examinte par spectrophotornetrie. La rtaction est nbgligeable comparte B celle des autres groupements. L'adtyl histidine et l'imidazsle ne rtagissent pas. Nous comparons la rkactivitt des groupements a-amints, e-aminb et celle d'autres rtsidus dans les proteines. Nous discutons en dktail l'ktat conformationnel des rtsidus N-terminaux des sCrine proteinases tel que montrk par leur rtactivitk. Nous concluons que I'acide nitreux rkagit de faqon prefkrentielle avec les rCsidus de '6s...

Biological Tyrosine Nitration: A Pathophysiological Function of Nitric Oxide and Reactive Oxygen Species

“…In the case of ␤-casein there was also a complete loss of tryptophan residues. These observations are in apparent agreement with the rate constants reported by Hofman and co-workers for the reaction of nitrite at pH 4.0 with amino acid residues (93). Tryptophan reacted considerably faster than either lysine or tyrosine.…”

“…Tryptophan reacted considerably faster than either lysine or tyrosine. The apparent pseudo-first order rate constant for the reaction of 0.1 M nitrite at pH 4.0 was 0.26, 2.5 ϫ 10 Ϫ5 , and 5 ϫ 10 Ϫ5 -10 Ϫ6 s Ϫ1 for ␣-N-acetyltryptophan, ␣-benzoyllysine, and ␣-N-acetyltyrosine, respectively (93). Moreover, if the protein contains free cysteine residues, reaction with acidified nitrite results in the formation of nitrosocysteine (94).…”

Deamination of lysine as a marker for nitrite-protein reactions

“…In some investigations (Knowles et al, 1974 and Small, 1938;Shields et al, 1959) the variable reactivity of some amino acid residues with nitrite was noted, but accompanying amino acid analyses were ambiguous. Losses of some amino acids were evident in the data but were not recognized as significant; new peaks were either overlooked (Kurosky and Hoffman, 1972) or not identified (Knowles et al, 1974; Natake and Ueda, 1986).…”

Effect of sodium chloride on some of the chemical reactions of sodium nitrite in cured meat

“…This might be because the added cysteine acted as a reducing agent and was more available to nitrite for reaction. The fact that nitrous acid reacted preferentially with surface residues (Kurosky and Hofmann, 1972) can be another explanation. When mixtures of 19 amino acids, which contained cystine instead of cysteine, were incubated with nitrite at 37°C, no decrease in nitrite was observed.…”

“…The alpha-amino group of amino acids, the epsilon-amino group of lysine, the ring nitrogen of tryptophan, and tyrosine could react with nitrous acid (Kurosky and Hofmann, 1972), thus depleting nitrite. Reac tion of tyrosine with nitrous acid has been shown by many workers (Philpot and Small, 1938;Means and Feeney, 1971; Bard and Townsend, 1971;Knowles, 1974;Woolford et al, 1976).…”