An exciting but challenging road ahead for computational enzyme design.

Recent work has demonstrated that computational enzyme design can generate active catalysts. Although the progress is encouraging, the future will be brighter for this new field if its current limitations and the challenges which must be overcome are as broadly understood as its promise. This essay compares the activities of de novo designed enzymes to those of naturally occurring enzymes and highlights the considerable challenges which must be overcome for computational design to produce enzymes with levels of activity similar to those of naturally occurring enzymes. Naturally occurring enzymes are exceptional catalysts. For example, arginine decarboxylase, alkaline phosphatase, and staphylococcal nuclease enhance the rates of the reactions they catalyze by more than 10 fold. The effective second order rate constants for naturally occurring enzymes are typically within three orders of magnitude of diffusion control. In contrast, most computationally designed enzymes to date provide rate enhancements of less than 10 and are more than six orders of magnitude from the diffusion limit. Furthermore, only a small fraction of computational designs have even these very modest levels of catalytic prowess; the majority have no detectable activity. A final caution is that the levels of activities that have been achieved are not much higher than those of catalytic antibodies developed 15–25 years ago. Clearly, computational enzyme design has a long way to go to consistently achieve native like levels of catalytic activity. Why are the activities of computationally designed enzymes and the overall design success rate so low? De novo design of enzyme catalysts requires models of ideal active sites that can catalyze the reaction of interest, and design methods that can create stable proteins which contain these sites. A design can fail at three different levels: first, the hypothesis represented by a proposed model of an ideal active site can be incorrect (perfect structural recapitulation in this case would not produce an active catalyst), second, the desired active site geometry may not be structurally realized by the actual design, and third, even with a correct active site description and perfect structural recapitulation, a designed enzyme can have little activity if the surrounding protein context, for example the long range electrostatics or dynamics, is not compatible with catalysis. Determining the reasons for the very low activity of designed enzymes could provide insight into important issues in both enzymology and protein design. Is the low activity because the original conception of the ideal active site was incorrect? Because the designed site was only in part realized? What is the influence of protein context—are protein elements not part of the original ideal active site impeding catalysis? Answering these questions will also provide the basis for iterative improvement of designed catalysts which will likely be critical to achieving high catalytic activity. Much work needs to be done to understand why current computationally designed enzymes are not *Correspondence to: David Baker, J557 Health Sciences Building, Box 357350, Seattle 98195, WA. E-mail: [email protected].