Effect of <img src='http://www.niscair.res.in/jinfo/smaller.gif' border=0>-amino acids on the functional activity and conformational stability of ribonuclease-A

Abstract

Using cytidine 2':3' cyclic monophosphate as a substrate, <i>K</i>_m and <i>k</i>_cat of ribonuclease-A in the presence of different concentrations of D-amino acids (Ala, Ser, Pro and Lys) and their L- isomers were measured at <i>p</i>H 6.0 and 25°C<i>. </i>These kinetic parameters remained unchanged in the presence and absence of D-and L-amino acids. This is the first experimental evidence showing that D-amino acids are compatible with the enzyme function. Values of <i>T</i>_m (midpoint of denaturation).Δ<i>H</i>_m<i> </i>(enthalpy change at <i>T</i>_m) and Δ <i>C</i>_p (constant-pressure heat capacity change) were also determined from the heat –induced denaturation curves of the protein. measured in the presence and absence of D- and L-isomers of an amino ac id at four

different <i>p</i>H values. It is shown for the first time that these thermodynamic parameters, within experimental errors, do not depend on the stereospecificity of an amino acid. Estimates of Δ<i>G</i>_D^o<i> </i>with the help of Gibbs-Helmoltz equation (Δ<i>G</i>_D^o = Δ<i>H</i>_m (1-298.15<i>/T</i>_m)<i> </i>- Δ<i>C</i>_p [(<i>T</i>_m- 298.15)<i> </i>+ 298.15 In (298.15/<i>T</i>_m)])using known values of <i>T</i>_m, Δ<i>Hm </i>and Δ<i>C</i>_p suggested that D- and L-amino acids are compatible with protein stability, for Δ<i>G</i>_D^o remained unchanged in the presence of amino acids.