Capturing the free energy of transition state stabilization: insights from the inhibition of mandelate racemase

Abstract

Mandelate racemase (MR) catalyses the Mg2+-dependent interconversion of (R)- and (S)-mandelate. To effect catalysis, MR stabilizes the altered substrate in the transition state (TS) by approximately 26 kcal mol–1(–ΔGtx), such that the upper limit of the virtual dissociation constant of the enzyme-TS complex is 2 × 10–19M. Designing TS analogue inhibitors that capture a significant amount of ΔGtxfor binding presents a challenge since there are a limited number of protein binding determinants that interact with the substrate and the structural simplicity of mandelate constrains the number of possible isostructural variations. Indeed, current intermediate/TS analogue inhibitors of MR capture less than or equal to 30% of ΔGtxbecause they fail to fully capitalize on electrostatic interactions with the metal ion, and the strength and number of all available electrostatic and H-bond interactions with binding determinants present at the TS. Surprisingly, phenylboronic acid (PBA), 2-formyl-PBA, andpara-chloro-PBA capture 31–38% of ΔGtx. The boronic acid group interacts with the Mg2+ion and multiple binding determinants that effect TS stabilization. Inhibitors capable of forming multiple interactions can exploit the cooperative interactions that contribute to optimum binding of the TS. Hence, maximizing interactions with multiple binding determinants is integral to effective TS analogue inhibitor design.This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’.