The zinc project was a bit more complicated. They tested many designs that didn’t include the water molecule that normally takes part in that reaction. These had a relatively low success rate, with only 14 out of 89 showing weak catalytic activity. When they modeled the ones that didn’t work, they found that the designs often let a water molecule block the site where reactions should take place.
So, they also tried designs where they placed a water molecule in the right location as an additional design constraint. These had a much higher success rate, with 35 out of 38 showing some catalytic activity. Again, however, their activity was considerably lower than the enzyme that was used as the starting point for the designs. (They did get one design with a better level of activity, but it wasn’t specific about where it cut other proteins.)
To find out whether this activity could be improved, the researchers identified amino acids that were in close proximity to the active site of the protease, but not actively involved with the catalysis. And they swapped in every alternative amino acid that wasn’t predicted to disrupt the structure of the design. They found some versions that were considerably more active; the best version they identified accelerated the cleavage of the peptide bond by a factor of 109 while remaining highly specific for the intended target protein sequence.
Similar things happened with the zinc-based protease. Their v2 version of their protease, which incorporated some of the random mutations, was 30 times more active than the original design it was based on. It also accelerated peptide-bond cleavage by a factor of 1010.
As a final demonstration of this approach’s promise, the researchers designed zinc proteases that could target proteins associated with neurological disorders, such as Alzheimer’s and frontotemporal dementia. Attempting to modify their existing design to target a new sequence didn’t work well, so they started the process over from the start and managed to find something that could cut the intended target in cells. For the researchers, this suggests that we might ultimately be able to influence the course of some genetic diseases by targeting the malformed proteins that cause them.