How do enzymes perform their job? How have they been trained by evolution to do it? And how do they communicate with their environment and their co-workers? These questions lie at the heart of understanding the complex network of biological reactions, which is essential for all processes of life. To answer these fundamental questions will be the goal of this proposal, which is aimed at developing a new line of interdisciplinary research combining single molecule experiments with methods allowing for the controlled manipulation of enzymatic activity such as allosteric regulation, site-directed mutagenesis, directed evolution and mechanical stress. Based on recent experimental and theoretical findings it is now widely accepted that enzymes are dynamic entities existing in different conformations and that the interconversion between these conformations results in observable fluctuations of the rate constants. Based on this picture allosteric regulation can be considered as a shift in the equilibrium between different conformations. During the evolution of new enzymes the newly emerging activity might be linked to a different conformation, which is then stabilized by mutations. To answer these key questions and to support the above hypotheses I propose to investigate the following aspects at the single enzyme level: 1. Analysis of the influence of mechanical manipulation on the catalytic activity 2. Analysis of the effect of mutations determining the dynamics of the active site on the catalytic performance 3. Analysis of variants evolved for higher activity with the goal of understanding to which extend the stabilization of a certain conformation contributes to the evolution process 4. Analysis of variants evolved to exhibit a different substrate specificity with the goal of understanding if evolution proceeds through multispecific intermediates characterized by a higher degree of conformational heterogeneity.