Beneficial mutations in pykF, which encodes pyruvate kinase, a major control-point enzyme of glycolysis, occurred in 12 independently evolved experimental populations of Escherichia coli propagated in a low glucose environment. We found that the evolved pykF mutations cause higher levels of its substrate, phosphoenolpyruvate (PEP), to be available at the start of each daily growth cycle. We hypothesize that higher PEP levels allow a quicker restart of the PEP-dependent glucose uptake system, providing a benefit by shortening lag times. Consistent with this hypothesis, competitive fitness assays show that selection favors pykF mutants most strongly in lag phase. This advantage is not general, being exclusive to resource environments in which PEP is necessary for sugar transport. We also found that the pykF mutations increased growth rates on gluconeogenic substrates, as expected if their changed function reduced consumption, and thus futile cycling, of PEP. Lastly, we manipulated expression of the ancestral pykF to test if fitness benefits could be reproduced through changes only in overall enzyme activity. Up- or down-regulating pykF did not mimic the fitness benefit conferred by mutant enzymes, suggesting that this benefit depended on a change in the enzyme's functional properties. Our study represents a rare comprehensive test of a proposed metabolic basis of the benefit of a naturally selected beneficial mutation. The mechanism of benefit is likely to be general and may be relevant to understanding the benefit conferred by changes in pyruvate kinase that are common in human cancer cells.