Practical considerations when using temperature to obtain rate constants and activation thermodynamics of enzymes with two catalytic steps: native and N460T-beta-galactosidase (E. coli) as examples.

Abstract

The values of the rate constants and the associated enthalpies and entropies of enzymes with two catalytic steps can be measured by determining the effects of temperature on the k (cat) values. Practical considerations that should be taken into account when doing this are presented. The narrow temperature range available with enzymes and the sensitivity of pH to temperature mean that special attention to detail must be taken and this study highlights the assiduousness needed. The necessity of conversion of apparent k (cat) to true k (cat) values when assays are done with products having pKa values near to the assay pH is shown and the importance of obtaining sufficient data is emphasized. Reasons that non-linear regression should be used to obtain the estimates of rate constants and activation thermodynamic parameters are given. Other precautions and recommendations are also presented. Results obtained by this method for native beta-galactosidase (E. coli) and for a beta-galactosidase in which a Thr was substituted for Asn-460 were analyzed to demonstrate the valuable mechanistic details of enzymes that can be obtained from studies of this type.