With early life likely to have existed in a hot environment, enzymes had to cope with an inherent drop in catalytic speed caused by lowered temperature. Here we characterize the molecular mechanisms underlying thermoadaptation of enzyme catalysis in adenylate kinase using ancestral sequence reconstruction spanning 3 billion years of evolution. We show that evolution solved the enzyme's key kinetic obstacle—how to maintain catalytic speed on a cooler Earth—by exploiting transition-state heat capacity. Tracing the evolution of enzyme activity and stability from the hot-start toward modern hyperthermophilic, mesophilic, and psychrophilic organisms illustrates active pressure versus passive drift in evolution on a molecular level, refutes the debated activity/stability trade-off, and suggests that the catalytic speed of adenylate kinase is an evolutionary driver for organismal fitness.

The advent of biocatalysts designed computationally and optimized by laboratory evolution provides an opportunity to explore molecular strategies for augmenting catalytic function. Applying a suite of NMR, crystallographic, and stopped-flow techniques to an enzyme designed for an elementary proton transfer reaction, we show how directed evolution gradually altered the conformational ensemble of the protein scaffold to populate a narrow, highly active conformational ensemble and achieve a nearly billionfold rate acceleration. Mutations acquired during optimization enabled global conformational changes, including high-energy backbone rearrangements, that cooperatively organized the catalytic base and oxyanion stabilizer, thus perfecting transition-state stabilization. Explicit sampling of conformational sub-states during design, and specifically stabilizing productive over all unproductive conformations, could speed up the development of protein catalysts for many chemical transformations.

Despite being the subject of intense effort and scrutiny, kinases have proven to be consistently challenging targets in inhibitor drug design. A key obstacle has been promiscuity and consequent adverse effects of drugs targeting the ATP binding site. Here we introduce an approach to controlling kinase activity by using monobodies that bind to the highly specific regulatory allosteric pocket of the oncoprotein Aurora A (AurA) kinase, thereby offering the potential for more specific kinase modulators. Strikingly, we identify a series of highly specific monobodies acting either as strong kinase inhibitors or activators via differential recognition of structural motifs in the allosteric pocket. X-ray crystal structures comparing AurA bound to activating vs inhibiting monobodies reveal the atomistic mechanism underlying allosteric modulation. The results reveal 3 major advantages of targeting allosteric vs orthosteric sites: extreme selectivity, ability to inhibit as well as activate, and avoidance of competing with ATP that is present at high concentrations in the cells. We envision that exploiting allosteric networks for inhibition or activation will provide a general, powerful pathway toward rational drug design.