RATES OF UNCATALYZED PEPTIDE-BOND HYDROLYSIS IN NEUTRAL SOLUTION AND THE TRANSITION-STATE AFFINITIES OF PROTEASES

To assess the relative proficiencies of enzymes that catalyze the hydr olysis of internal and C-terminal peptide bonds, the rates of the corr esponding nonenzymatic reactions were examined at elevated temperature s in sealed quartz tubes, yielding linear Arrhenius plots. The results indicate that in neutral solution at 25 degrees C, peptide bonds are hydrolyzed with half-times of approximately 500 years for the C-termin al bond of acetylglycylglycine, 600 years for the internal peptide bon d of acetylglycylglycine N-methylamide, and 350 years for the dipeptid e glycylglycine. These reactions, insensitive to changing pH or ionic strength, appear to represent uncatalyzed attack by water on the pepti de bond. Comparison of rate constants indicates very strong binding of the altered substrate in the transition states for the corresponding enzyme reactions, K-alpha attaining a value of less than 10(-17) M in carboxypeptidase B. The half-life of the N-terminal peptide bond in gl ycylglycine N-methylamide, whose hydrolysis might have provided a refe rence for assessing the catalytic proficiency of an aminopeptidase, co uld not be determined because this compound undergoes relatively rapid intramolecular displacement to form diketopiperazine (t(1/2) similar to 35 days at pH 7 and 37 degrees C). The speed of this latter process suggests an evolutionary rationale for posttranslational N-acetylatio n of proteins in higher organisms, as a protection against rapid degra dation.