Genetic Perturbation Alters Functional Substates in Alkaline Phosphatase

Enzymes inherently exhibit molecule-to-molecule heterogeneity
in
their conformational and functional states, which is considered to
be a key to the evolution of new functions. Single-molecule enzyme
assays enable us to directly observe such multiple functional states
or functional substates. Here, we quantitatively analyzed functional
substates in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase by employing
a high-throughput single-molecule assay with a femtoliter reactor
array device. Interestingly, many mutant enzymes exhibited significantly
heterogeneous functional substates with various types, while the wild-type
enzyme showed a highly homogeneous substate. We identified a correlation
between the degree of functional substates and the level of improvement
in promiscuous activities. Our work provides much comprehensive evidence
that the functional substates can be easily altered by mutations,
and the evolution toward a new catalytic activity may involve the
modulation of the functional substates.