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Evolution of new enzymatic activities is believed to require a period of gene sharing in which a single enzyme must serve both its original function and a new function that has become advantageous to the organism. Subsequent gene duplication allows one copy to maintain the original function, while the other diverges to optimize the new function. The physiological impact of gene sharing and the constraints imposed by the need to maintain the original activity during the early stages of evolution of a new activity have not been addressed experimentally. We report here an investigation of the evolution of a new activity under circumstances in which both the original and the new activity are critical for growth. Glutamylphosphate reductase (ProA) has a very low promiscuous activity with N-acetylglutamylphosphate, the normal substrate for ArgC (N-acetylglutamylphosphate reductase). A mutation that changes Glu-383 to Ala increases the promiscuous activity by 12-fold but decreases the original activity by 2,800-fold. The impairment in Pro and Arg synthesis results in 14-fold overexpression of E383A ProA, providing sufficient N-acetylglutamylphosphate reductase activity to allow a strain lacking ArgC to grow on glucose. Thus, reaching the threshold level of NAGP reductase activity required for survival required both a structural mutation and overexpression of the enzyme. Notably, overexpression does not require a mutation in the regulatory region of the protein; amino acid limitation attributable to the poor catalytic abilities of E383A ProA causes a physiological response that results in overexpression. catalytic promiscuity | molecular evolution | ProA | recruitment