Kinetics of carboxymethylation of histidine hydantoin

Abstract

The reaction of the imidazole group of histidine hydantoin with bromoacetate was studied as a model for carboxymethylation of histidine residues in proteins. pK values of 6.4 and 9.1 (25 degrees C) and apparent heats of ionization of 7.8 and 8.7 kcal/mol were determined for the imidazole and hydantoin rings, respectively. At pH values corresponding to the isoelectric points for histidine hydantoin, the rates of carboxymethylation at 12, 25, 37, and 50 degrees C were determined; the modified hydantoins were hydrolyzed to the corresponding histidine derivatives for quantitative amino acid analysis. At pH 7.72 and 25 degrees C, the imidazole tele-N was alkylated (k = 3.9 X 10(-5) M-1 s-1) twice as fast as the pros-N. The monocarboxymethyl derivatives were carboxymethylated at the same rate at the pros-N (k = 2.1 X 10(-5) M-1 s-1) but 3 times faster at the tele-N (k = 11 X 10(-5) M-1 s-1). The enthalpies of activation determined for carboxymethylation of the imidazole ring and its monocarboxymethyl derivatives were similar (15.9 +/- 0.7 kcal/mol). delta S for the four carboxymethylations was -25 +/- 2 eu. The electrostatic component of delta S (delta S es) was calculated from the influence of the dielectric constant on the reaction rate at 25 degrees C. delta S es was slightly negative (-4 +/- 1 eu) for mono- or dicarboxymethylations, indicating some charge separation in the transition state. The nonelectrostatic entropy of activation was -21 +/- 2 eu for all four carboxymethylations.

Transition-state analysis of the facilitated alkylation of ribonuclease A by bromoacetate

“…Double-reciprocal plots of the rate of inactivation of RNase as a function of bromoacetate concentration were linear at all concentrations of dioxane, and the slopes, but not the intercepts, of these plots decreased slightly with dioxane concentration. The effect of dioxane on k}/K¡ was consistent with a value for A5*es of 3.7 ± 0.1 cal/(deg mol), calculated as described before (Lennette & Plapp, 1979), and is associated with the bimolecular alkylation of the free enzyme, perhaps reflecting electrostatic binding of the negatively charged bromoacetate to an imidazolium group. However, Maurel & Douzou (1975) established the compensation temperature for cationic acids (including His-46 in trypsin) as 37 ± 5 °C.…”

“…The RNase samples were desalted on a column of Sephadex G-25 equilibrated with 0.1 M HCOOH, oxidized with HCOOOH (Moore, 1963), hydrolyzed with 6 M HC1 for 46 h (Moore & Stein, 1963), and analyzed as described previously (Lennette & Plapp, 1979). The ratio of His(ffCm)/His(rCm) at the various temperatures gave a mean value of 4.4 ±0.1, and with various concentrations of dioxane, it was 5.4 ± 0.6.…”

Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications

“…Early His modifications were achieved by using bromoacetic acid and bromoacetate. ,, Two nitrogen atoms on the imidazole are potential nucleophilic sites for modification. As halogen atoms are good leaving groups, many α-halo carboxylic acids and amides were developed and utilized in His labeling, including but not limited to bromoacetate, ,− chloroacetate, ,, iodoacetate, ,, bromoacetamide, and IAM . Although effective, these reagents soon fell into disuse, primarily due to their lack in specificity.…”

Kinetic analysis of protein modification reactions at equilibrium

“…Examples of stoichiometric protein modification co-operativity are: (1) the carbamoylation of the four terminal valine residues of human haemoglobin (positive co-operativity) and (2) the photo-oxidation of the methionine residues of phosphoglucomutase (negative co-operativity) (Rakitzis, 1983a(Rakitzis, , 1984a. Examples of site-oriented co-operativity are: (1) the carboxymethylation of the histidine hydantoin derivative (Lennette & Plapp, 1979;Rakitzis, 1983c) and (2) the carboxymethylation of either His-119 or His-12, never both, of ribonuclease A (Heinrikson et al, 1965). A special case of negative modification co-operativity has been termed 'half-of-the sites reactivity' (Stallcup & Koshland, 1973a,b;Schwendimann et al, 1976).…”