The wealth of distinct enzymatic functions found in nature is impressive and the on-going evolutionary divergence of enzymatic functions continues to generate new and efficient catalysts, which can be seen through the recent emergence of enzymes able to degrade xenobiotics. However, recreating such processes in the laboratory has been met with only moderate success. What are the factors that lead to suboptimal research outputs? In this review, we discuss constraints on enzyme evolution, which can restrict evolutionary trajectories and lead to evolutionary dead-ends. We highlight recent studies that have used experimental evolution to mimic different aspects of enzymatic adaptation under simple, controlled settings to shed light on evolutionary dynamics and constraints. A better understanding of these constraints will lead to the development of more efficient strategies for directed evolution and enzyme engineering. J. Exp. Zool. (Mol. Dev. Evol.) 322B: 468–487, 2014. © 2014 Wiley Periodicals, Inc.