A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid Isomerase.

ABSTRACT: The vibrational Stark effect (VSE) has been used to measure the electric field in the active site of ketosteroid isomerase (KSI). These measured fields correlate with ΔG(⧧) in a series of conventional mutants, yielding an estimate for the electrostatic contribution to catalysis (Fried et al. Science 2014, 346, 1510-1513). In this work we test this result with much more conservative variants in which individual Tyr residues in the active site are replaced by 3-chlorotyrosine via amber suppression. The electric fields sensed at the position of the carbonyl bond involved in charge displacement during catalysis were characterized using the VSE, where the field sensitivity has been calibrated by vibrational Stark spectroscopy, solvatochromism, and MD simulations. A linear relationship is observ

SUBMITTER: Wu Y 

PROVIDER: S-EPMC5063566 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature