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

Abstract

Bromoacetate reacts with histidine residues 12 and 119 at the active site of bovine pancreatic ribonuclease (RNase) much more rapidly than with free histidine. The mechanism of this facilitated alkylation was investigated by studying the dependence of the reaction on temperature and pH. RNase was treated with bromoacetate under pseudofirst-order conditions at 12, 25, 37, and 50 O C . The rate of inactivation of the enzyme showed a hyperbolic dependence on bromoacetate concentration, indicating formation of an enzyme-bromoacetate complex (K, = 41 m M at pH 5.5 and 25 "C). Two groups, one of which must be unprotonated and the other protonated, are required for carboxymethylation of RNase by bromoacetate. At 25 OC, the free enzyme exhibits macroscopic pK values of 4.7 and 6.3, and the enzymebromoacetate complex has pK values of 5.8 and 7.4. The ratio of products [N"-(carboxymethy1)histidine-1 19 RNase to N'-(carboxymethy1)histidine-12 RNase] formed in the reaction was 4.4 and was independent of temperature. Calculations based on this ratio and the microscopic pK values of histidines-1 19 and -12 determined by N M R titration suggest that the pH-independent alkylation of histidine-1 19 is about 8 times

A bait and switch hapten strategy generates catalytic antibodies for phosphodiester hydrolysis

“…X-ray diffraction (15), chemical modification (16), and pH-rate studies (17) are all consistent with an enzymatic reaction mechanism in which the rate-limiting transition state for RNA cleavage is similar to that shown in Fig. 2.…”

Enthalpic and entropic determinants for the specificity of alkylation of the histidine‐12 residue of ribonuclease A by four bromoacetamido nucleoside affinity labels and bromoacetamide

“…The constants, E, and k,', are the pH-independent alkylation rate constants at unprotonated and protonated His 12. The kinetic scheme is simplified by assuming that E3' = 0, K, = K,', K, = K,' and acetate ion binds negligibly to the unprotonated form of the free enzyme (9) and not all to the RNase A * bromoacetamido nucleoside complexes, E -AL and EH+ -AL. K, = K,' implies that binding of the nucleoside does not alter the ionization constant for His12 relative to its value in the free enzyme.…”

“…Second, acetate binds negligibly to the unprotonated Hisl 2 form of RNase A. Between 12" and 37", the ratio of the limiting dissociation constants of the RNase Abromoacetate complex at high pH to that at low pH values exceeds 100 (9). Third, bromoacetate binding to RNase A is a good model for acetate binding.…”

Kinetics of protein modification reactions

“…If two nonidentical acidic groups react with the modifying agent, the concentration of the reactive, unprotonated or dissociated, form is given by the Michaelis pH functions (Cornish-Bowden, 1976), and plots of kapp. verus pH are bell-shaped (Lennette & Plapp, 1979). A study of the pH dependence of covalent modification by two-protonic state electrophiles has been presented by Brocklehurst (1982).…”

“…The temperature dependence of reaction rate constants yields the values for the enthalpy, as well as for the entropy change, of the formation of the activated complex of the reaction under study (Frost & Pearson, 1961;Gutfreund, 1972;Laidler & Bunting, 1973). It has been pointed out by Lennette & Plapp (1979) that, in contrast with studies of enzyme catalysis, the thermodynamic parameters for the rate constants of the reaction of modifying agents with proteins can always be compared with the parameters of the reaction of the same modifying agent with small molecules containing the same functional groups as those with which they react in the intact protein. 'Active-site directed reagents resemble substrates in their behaviour towards enzymes: they bind to the active site and their rates of reaction with the enzyme are facilitated, presumably by one or more catalytic factors.…”