TRANSITION-STATE ANALYSIS OF THE FACILITATED ALKYLATION OF RIBONUCLEASE-A BY BROMOACETATE

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 pseudo-first-order conditions at 12, 25, 37, and 50 °C. The rate of inactivation of the enzyme showed a hyperbolic dependence on bromoacetate concentration, indicating formation of an enzyme-bromoacetate complex (K1, = 41 mM 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 °C, the free enzyme exhibits macroscopic pK values of 4.7 and 6.3, and the enzyme-bromoacetate complex has pK values of 5.8 and 7.4. The ratio of products [Nπ-(carboxymethyl) histidine-l19 RNase to Nπ-(carboxymethyl)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-119 and -12 determined by NMR titration suggest that the pH-independent alkylation of histidine-119 is about 8 times faster than that of histidine-12. The pH-independent rate of alkylation of RNase is 440 times that of histidine hydantoin at 25 °C. Significantly, ΔH‡ for carboxymethylation of either histidine residue was 11 ± 2 kcal/mol, compared to 16.5 ± 0.4 kcal/mol for alkylation of histidine hydantoin. Values of TΔS‡ for the pseudobimolecular reaction of RNase and bromoacetate (-9 ± 2 kcal/mol) and for histidine hydantoin (-7.1 ± 0.3 kcal/mol) were similar. The carboxymethylation of RNase appears to be greatly facilitated by enthalpic factors, which increase the inherent reactivities of the histidine residues, rather than by entropic factors, such as binding and orientation. (However, differential solvation effects may have masked the intrinsic entropy changes that can facilitate the enzymatic reaction by reducing the loss of translational and rotational entropy in forming the transition state.) There is little or no electrostatic component of ΔS‡ associated with the unimolecular reaction of the enzyme-bromoacetate complex, suggesting that charge separation is stabilized in the transition state. The microenvironments of the histidine residues in the RNase-bromoacetate complex apparently confer unusual properties on the imidazole groups. © 1979, American Chemical Society. All rights reserved.