Rates of Uncatalyzed Peptide Bond Hydrolysis in Neutral Solution and the Transition State Affinities of Proteases

Abstract

To assess the relative proficiencies of enzymes that catalyze the hydrolysis of internal and C-terminal peptide bonds, the rates of the corresponding nonenzymatic reactions were examined at elevated temperatures in sealed quartz tubes, yielding linear Arrhenius plots. The results indicate that in neutral solution at 25 °C, peptide bonds are hydrolyzed with half-times of approximately 500 years for the C-terminal bond of acetylglycylglycine, 600 years for the internal peptide bond of acetylglycylglycine N-methylamide, and 350 years for the dipeptide glycylglycine. These reactions, insensitive to changing pH or ionic strength, appear to represent uncatalyzed attack by water on the peptide bond. Comparison of rate constants indicates very strong binding of the altered substrate in the transition states for the corresponding enzyme reactions, K tx attaining a value of less than 10-17 M in carboxypeptidase B. The half-life of the N-terminal peptide bond in glycylglycine N-methylamide, whose hydrolysis might have provided a reference for assessing the catalytic proficiency of an aminopeptidase, could not be determined because this compound undergoes relatively rapid intramolecular displacement to form diketopiperazine (t 1/2 ∼ 35 days at pH 7 and 37 °C). The speed of this latter process suggests an evolutionary rationale for posttranslational N-acetylation of proteins in higher organisms, as a protection against rapid degradation.

Binding Affinities of Host–Guest, Protein–Ligand, and Protein–Transition‐State Complexes

“…Wolfenden and co-workers have compiled and measured many of these, and they provide most of the data that allow calculation of the K tx À1 values plotted in Figure 21. [40] The average value of K tx À1 for enzymes of 10 16.0AE4.0 m À1 corresponds to an average free energy of binding of 22 kcal mol À1 , but can range up to 32 kcal mol À1 . [34b, 40] The value of k cat.…”

“…Plot of the number of complexes formed between enzymes and transition states with values of Àlg K tx falling within a particular range (K a = 10 16.0AE4.0 m À1 ). [40] as K M K tx À1 . From our previous discussion, the average value of K M is 10 À3.7 m. Therefore, the average value of k cat.…”

Metal assisted peptide bond hydrolysis: Chemistry, biotechnology and toxicological implications

“…Its half-life is estimated at 350-600 years at 25 • C and neutral pH [5]. This remarkable kinetic stability is required for its function, but presents a challenge when there is a physiological need to break it.…”

Spin Equilibria in Monomeric Manganocenes: Solid-State Magnetic and EXAFS Studies

“…4 Similarly, enzymatic peptidases are able to cleave peptide bonds quite efficiently with rate accelerations of up to 10 10 -10 12 when compared to the background hydrolysis under physiological conditions. [5][6][7] Inspired by this enzyme-mediated reactivity, synthetic chemists have sought to both explain and emulate such reactivity in synthetic molecular assemblies. Despite the intense study of enzymatic selectivity and efficiency, a complete understanding of how enzymes are able to achieve such heightened reactivity remains elusive.…”