Orotic acid is decarboxylated with a half-time (t1/2) of 78 million years in neutral aqueous solution at room temperature, as indicated by reactions in quartz tubes at elevated temperatures. Spontaneous hydrolysis of phosphodiester bonds, such as those present in the backbone of DNA, proceeds even more slowly at high temperatures, but the heat of activation is less positive, so that dimethyl phosphate is hydrolyzed with a t1/2 of 130,000 years in neutral solution at room temperature. These values extend the known range of spontaneous rate constants for reactions that are also susceptible to catalysis by enzymes to more than 14 orders of magnitude. Values of the second-order rate constant kcat/Km for the corresponding enzyme reactions are confined to a range of only 600-fold, in contrast. Orotidine 5'-phosphate decarboxylase, an extremely proficient enzyme, enhances the rate of reaction by a factor of 10(17) and is estimated to bind the altered substrate in the transition state with a dissociation constant of less than 5 x 10(-24) M.
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.
Peptide bonds interact so strongly with water that even a modest difference between the free energies of solvation of their cis and trans isomers could have a significant bearing on protein structure. However, proton magnetic resonance studies at high dilution in deuteriated solvents show that N-methylformamide exists as the cis isomer to the extent of 8% in water, 10.3% in chloroform, 8.8% in benzene, and 9.2% in cyclohexane. Integrated intensities of proton and carbon resonances show that N-methylacetamide exists as the cis isomer to the extent of only 1.5% in water, not changing much in nonpolar solvents. Quantum mechanical calculations using the 6-31G basis set reproduce these relative abundances with reasonable accuracy and show that there is little difference between the dipole moments of the cis and trans isomers, for either amide. The remarkable insensitivity of cis/trans equilibria to the solvent environment and the heavy preponderance of trans isomers regardless of the polarity of the surroundings (ca. 98.5% for N-methylacetamide, whose properties may resemble those of a typical peptide bond) accord with the overwhelming preference of peptide bonds for the trans configuration that is consistently observed in the three-dimensional structures of globular proteins.
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