Abstract:

Why highly stable proteins are not commonly observed in mesophilic organisms is an important evolutionary question. It has been suggested that high stability has a fitness cost in the form of loss of activity. However, this hypothesis has not yet been experimentally demonstrated. Here, we use an essential bacterial enzyme adenylate kinase (Adk) to explore this hypothesis and show that, as Adk’s stability increases, one of its own substrates inhibits its activity. Bacterial strains carrying such stable Adks show substantial fitness defects, which can be mapped to the loss of Adk activity due to substrate inhibition. Overall, our study adds “substrate inhibition” to the “toolbox” that is used to rationalize the stability distribution of proteins.Proteins are only moderately stable. It has long been debated whether this narrow range of stabilities is solely a result of neutral drift toward lower stability or purifying selection against excess stability—for which no experimental evidence was found so far—is also at work. Here, we show that mutations outside the active site in the essential Escherichia coli enzyme adenylate kinase (Adk) result in a stability-dependent increase in substrate inhibition by AMP, thereby impairing overall enzyme activity at high stability. Such inhibition caused substantial fitness defects not only in the presence of excess substrate but also under physiological conditions. In the latter case, substrate inhibition caused differential accumulation of AMP in the stationary phase for the inhibition-prone mutants. Furthermore, we show that changes in flux through Adk could accurately describe the variation in fitness effects. Taken together, these data suggest that selection against substrate inhibition and hence excess stability may be an important factor determining stability observed for modern-day Adk.

Website