Title: Substrate inhibition imposes fitness penalty at high protein stability,
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.