Substrate inhibition imposes fitness penalty at high protein stability

TL;DR

This study demonstrates that high protein stability can lead to substrate inhibition, impairing enzyme activity and fitness, suggesting natural selection favors moderate stability to avoid such penalties.

Contribution

It provides experimental evidence that substrate inhibition at high stability influences protein evolution, explaining the narrow stability range of modern proteins.

Findings

Mutations outside active site increase substrate inhibition at high stability.

Substrate inhibition impairs enzyme activity and reduces fitness.

Flux variations correlate with fitness effects.

Abstract

Proteins are only moderately stable. It has long been debated whether this narrow range of stabilities is solely a result of neutral drift towards lower stability or purifying selection against excess stability is also at work - for which no experimental evidence was found so far. Here we show that mutations outside the active site in the essential E. coli enzyme adenylate kinase result in 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. Further, 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 have resulted in a narrow range of optimal stability observed for modern proteins.