Temperature effects on S 1- and S 1′-enantioselectivity of α-chymotrypsin

Abstract

The temperature dependence of E (enantiomeric ratio or enantioselectivity, a quantitative measure for enzyme stereospecificity) has been studied for the α-chymotrypsin catalysed hydrolysis of the enantiomeric N-Boc- l/ d-TyrOMe, l/ d-TyrOMe, Ac- l/ d-PhgOMe, l/ d-PhgOMe and for the kinetically controlled synthesis of the diastereomeric dipeptides N-Ac- l-Tyr- l/ d-ArgNH 2 and N-Ac- l-Tyr- l/ d-ValNH 2. The results show that the S 1- and S 1′-enantioselectivity can be modulated by the temperature (3–15 fold for the studied substrates in the range 5–45°C). For l/ d-PhgOMe a reversal in stereospecificity was found in this temperature interval. For the studied substrates both an increase or decrease of the enantiomeric ratio with increasing temperature was observed. For these processes the following relation for the temperature dependence of E has been derived ln E= ln(k l /k d )=−( ΔΔH #− ΔΔH b )/RT+( ΔΔS #− ΔΔS b )/R where k l and k d are apparent second order rate constants for the reactions with the l- and d-enantiomers, respectively. ΔΔ denotes the differences between the thermodynamic parameters for transformation of the enantiomeric substrates. The subscript b applies for the binding of the substrate or the nucleophile and the superscript # for the formation of the transition state of the enzyme acylation or deacylation. For the studied processes either the enthalpy (ΔΔ H #−ΔΔ H b) or the entropy (ΔΔ S #−ΔΔ S b) term was found to control the discrimination. Thus, the enantioselectivity decreases or increases with temperature, respectively. The influence of ground-state interactions and transition-state stabilisation on enzyme enantioselectivity has been discussed.