Solute transfer between intramolecular polar and non-polar phases as a possible rate-limiting step in chymotrypsin deacylation

Abstract

Equations predicting the relative rate of solute transfer from a polar to a non-polar phase and from a non-polar to a polar phase are derived on the basis of a model which assumes that the solute becomes partially dissociated from both phases during transfer. It is further assumed that the rate-limiting step in the deacylation of α-chymotrypsin is a transfer between phases rather than the hydrolysis itself, or alternatively, that the rate of hydrolysis depends on the rate of transfer and the equations are applied to the rates of deacylation of the acetyl through the caproyl derivatives measured by Cane & Wetlaufer (1966) at pH 8 · 1. For a rate-limiting transfer from a non-polar to a polar phase the following relationship would be expected: log ( KC) = γ log ( PC) + constant; whereas for a transfer from a polar to a non-polar phase: log ( K C ) = γ log ( P C ) + constant ; where K is the first-order constant for deacylation, Ci s the non-polar: polar partition coefficient for the free acid derived from the acyl group, P is the vapor pressure of the free acid, and γ is a fraction between 0 and 1 · 0 which is related to the extent of dissociation of the acid from both phases during transfer. (A γ of 1 · 0 implies complete dissociation from both phases.) Using the benzene: water partition coefficient for C, the rate constants are found to be consistent with a rate-limiting transfer from a polar to a non-polar phase; for the C 2 through C 4 acids or acyl groups, γ = 0 · 5, whereas for the C 5 and C 6 groups γ is 0 · 1 to 0 · 2, indicating that a definite change in the mode of transfer occurs between C 4 and C 5 such that the shorter-chain groups lose contact with the two phases to a greater extent during transfer. Indole is found to increase γ for all derivatives, suggesting that indole tends to pull the two phases apart. Since the experimental procedure used to determine the rates of deacylation would measure the total hydrolysis rather than the release of the free acids from the enzyme, the foregoing deductions would imply that a rate-limiting transfer of the attached acyl group occurs prior to hydrolysis, and that the hydrolysis would ensue relatively rapidly once the configuration of the acyl group had been altered by the transfer. Inasmuch as the acyl groups considered represent poor substrates for chymotrypsin, it is also possible that the hydrolysis rather than the transfer becomes rate-limiting for the good substrates. The increase in the ΔS ∗ for deacylation between C 4 and C 5 found by Cane & Wetlaufer (1966) as well as the “better binding: better reaction” postulate of Knowles (1965) are considered in light of these findings.