Hydrogen bonding in enzymatic catalysis analysed by protein engineering

Abstract

Two historic publications have suggested that enzymes can utilize the binding energy with their substrates to increase the rate of chemical catalysis—a concept termed strain^1,2. Specifically, the structure of an enzyme was described as complementary to the structure of the transition state of its substrate rather than to that of the unreacted substrate, so that the substrate would tend to be distorted on binding to the enzyme. The current understanding of strain does not require such distortion. Instead, it is thought that there are groups on the enzyme which interact better with the transition state than with the unaltered substrate, an arrangement known as differential binding of substrates and transition states³. The improvement in binding energy caused by going from substrate to transition state lowers the activation energy of the reaction and so increases the catalytic rate. Here we report direct evidence in support of this notion. Rapid reaction kinetics on mutants of a tyrosyl-transfer RNA synthetase produced by protein engineering show that certain hydrogen-bonding side chains some distance from the reacting bonds of the substrate bind to ATP preferentially in the transition state of the reaction rather than in the unreacted state. In this way, the binding energies of the interactions are used primarily to lower the apparent activation energy of the chemical steps rather than to enhance binding.