The free energy–reaction coordinate profile for α‐chymotryptic hydrolysis of a series of N‐acetyl‐α‐L‐amino acid methyl esters

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Abstract
The effect of added nucleophiles (methanol and 1,4‐butanediol) on the steady‐state kinetics of α‐chymotryptic hydrolysis of a series of N‐acetyl‐L‐amino acid methyl esters, R‐CH(NHCOCH3)C(O)OCH3, has been studied. As a result, the rate and equilibrium constants of the ‘elementary’ steps of the enzyme process have been determined.It has also been demonstrated how the free energy–reaction coordinate profile changes if the structure (the size of the hydrocarbon chain) of the ‘chemically inert’ substrate fragment R is varied. The effects observed can be described by the following equation: equation image where ΔGs and ΔGa are the free energies of formation of metastable intermediates, i.e., the enzyme–substrate complex and the acylenzyme, respectively, ΔG2≠ and ΔG3≠ are the free energies of activation for the chemical steps, i.e., enzyme acylation and acylenzyme hydrolysis, respectively; and ΔGtrans(R) is the free energy of transfer of substrate group R from water into a nonaqueous solvent.To explain the results obtained, a mechanism for enzyme–substrate interaction is suggested according to which the potential free energy of sorption of substrate group R on the enzyme is 2 ΔGtrans(R). Such a high gain in the free energy of hydrophobic interaction may only be realized if (a) in the free enzyme the sorption region has a thermodynamically unfavorable contact with the aqueous medium, and (b) water is forced out of the active center as a result of the hydrophobic interaction of substrate group R with the enzyme. Such a model is in agreement with the published x‐ray data on the structure of the crystalline enzyme.The kinetic experiment has proved that not all the potential free energy of sorption is realized as binding force. Thus the true free energy of the binding of substrate group R with the protein does not exceed half the maximum value, both in the enzyme–substrate complex and acylenzyme.