Vibrational Stark Effects in the Active Site of Ketosteroid Isomerase Point to Large Electric Fields Driving Chemical Catalysis

Abstract

Enzymes are extraordinary catalysts that actuate nearly all biomolecular processes with speed and specificity. Nevertheless, the physical origins of enzymes’ catalytic power remain elusive despite investigations of many enzymes’ mechanisms over the last half-century. Ketosteroid isomerase (KSI) is a small and proficient enzyme - accelerating an isomerization reaction ∼1 trillion-fold over its intrinsic rate in water - that has been employed as a test system to examine the catalytic strategies at Nature's disposal. Electrostatic interactions are broadly purported to play an essential role in catalysis, but this proposal has yet to be experimentally tested in a quantitative fashion. Toward this end, vibrational Stark effect spectroscopy provides a toolkit to measure electric fields in biomolecular environments. By measuring the vibrational Stark effect of an intrinsic probe that mimics the reactive species of KSI's catalytic cycle, we found that KSI's active site focuses an extremely large electric field onto the scissile bond, potentially enabling its speedy chemical conversion through electrostatic interactions. Moreover, we observed a strong correlation between active site electric field and catalytic power across several KSI mutants. These studies are building toward a highly reductionist picture of KSI's catalysis and possibly enzyme catalysis in general.