Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Go big or you'll get lost

Rational mutagenesis is a common approach to investigating or engineering enzyme function in vitro, but the ease with which one can manipulate protein sequences belies many pitfalls in connecting sparse activity data to an enzyme's true functional landscape. Using a high-throughput platform, Markin et al. expressed, purified, and performed an array of kinetic measurements on a target esterase, collecting data from >1000 mutations spanning the entire protein (see the Perspective by Baumer and Whitehead). Protein misfolding into an inactive state, rather than decreased equilibrium stability, was a crucial factor in negatively affected variants spread throughout the protein. When combined with prior mechanistic understanding and structures, four “functional components” help to rationalize the otherwise complex spatial pattern of effects of mutations on different aspects of enzyme function, all of which would be invisible from mutagenesis of just a few residues.

Science , abf8761, this issue p. eabf8761 ; see also abj8346, p. 391