Relationships between apparent binding energies measured in site-directed mutagenesis experiments and energetics of binding and catalysis.
The use of binding energy in molecular recognition and enzyme catalysis is currently being probed by experiments on engineered proteins. The interaction energy of an individual side chain with a substrate may be quantified by comparing the binding and rate constants for wild-type enzyme with those for a mutant in which the side chain has been truncated. An apparent binding energy delta Gapp is obtained. The physical significance of delta Gapp is analyzed with particular reference to hydrogen bonding where one partner in the bond is deleted by mutagenesis. The following conclusions have been drawn for situations where mutagenesis does not unduly perturb the structure of the protein. delta Gapp is always a measurement of specificity of binding and catalysis. But, it does not generally measure the incremental binding energy of the hydrogen bond delta Gbind. The discrepancy between delta Gapp and delta Gbind is especially large when mutation leaves a charged donor or acceptor unpaired. Here, delta Gapp overestimates delta Gbind by possibly several kilocalories per mole. On the other hand, changes in delta Gapp (delta delta Gapp) as a reaction proceeds through its intermediates and transition states are particularly amenable to simple analysis. It is shown that delta delta Gapp can measure changes in delta Gbind (delta delta Gbind). For example, if there is a change in the energy of an individual bond on going from one state to the next, then delta delta Gapp = delta delta Gbind.(ABSTRACT TRUNCATED AT 250 WORDS)
Fersht
A R
AR
Department of Chemistry, Imperial College of Science and Technology, London, U.K.
D002151:Calorimetry; D004798:Enzymes / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D008962:Models, Theoretical; D009154:Mutation; D011485:Protein Binding; D013816:Thermodynamics
D004798:Enzymes