Substitution for Asn460 cripples β-galactosidase (Escherichia coli) by increasing substrate affinity and decreasing transition state stability.

Substrate initially binds to β-galactosidase (Escherichia coli) at a 'shallow' site. It then moves ∼3Å to a 'deep' site and the transition state forms. Asn460 interacts in both sites, forming a water bridge interaction with the O3 hydroxyl of the galactosyl moiety in the shallow site and a direct H-bond with the O2 hydroxyl of the transition state in the deep site.

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.

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.