A role for nuclear tunneling and protein dynamics in enzyme catalysis

A current topic of intensive research concerns the role of protein dynamics in enzyme catalysis. The measurement of kinetic isotope effects has emerged as a powerful probe of protein motions relevant to catalysis in enzymes that catalyze hydrogen transfer by a tunneling mechanism (Chapter 1). We have explored the role of protein motion in catalysis in a thermophilic bacterial alcohol dehydrogenase, and a psychrophilic homologue, which shares 61% sequence identity. Mutagenesis of a conserved hydrophobic residue in the cofactor binding domain of the thermophilic enzyme leads to two important observations (Chapter 2). First, it was seen that, upon mutation, the temperature dependence of the kinetic isotope effect was greatly increased above 30°C, in a temperature range where the wild type enzyme shows essentially temperature independent kinetic isotope effects. This is interpreted to mean that the hydrogen donor and acceptor undergo distance sampling upon the introduction of a packing defect at the active site of the enzyme. Second, below 30°C, Arrhenius prefactors as large as 1023 sec-1 are observed. This is interpreted as direct evidence of a class of motion, called conformational sampling (pre-organization) that is distinct from the process of reorganization, and which may involve regions of the protein distal to the active site. Mutagenesis of a conserved active site tryptophan (W87) in the thermophilic alcohol dehydrogenase (Chapter 3) leads to cold lability, and a loss of the Arrhenius break. This has been interpreted as a retention of the flexibility attained only above 30°C with the wild type enzyme. Because W87 is not located at the subunit interface, the effects of mutation at this position appear to be communicated to the protein surface. Finally, mutation of a single residue in the psychrophilic homologue of the thermophilic alcohol dehydrogenase confers thermostability (Chapter 4). The data are integrated into a general discussion of the role of protein motion in enzyme catalysis, and future research directions are proposed (Chapter 5).