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

Abstract

Mandelate racemase (MR) catalyses the Mg 2+ -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 (–Δ G tx ), such that the upper limit of the virtual dissociation constant of the enzyme-TS complex is 2 × 10 –19 M. Designing TS analogue inhibitors that capture a significant amount of Δ G tx for 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 Δ G tx because 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, and para -chloro-PBA capture 31–38% of Δ G tx . The boronic acid group interacts with the Mg 2+ 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’.

Reactivity and mechanism in chemical and synthetic biology

“…To date, however, few inhibitors with even picomolar affinities have been reported. Bearne [21] reviews the problems associated with designing these socalled transition state analogues, using mandelate racemase as a model system (figure 2). His analysis demonstrates the importance of maximizing the number of interactions within the active site in addition to ensuring that the electrostatic and steric properties of the molecule are similar to those calculated for the transition state.…”

A Phenylboronic Acid-Based Transition State Analogue Yields Nanomolar Inhibition of Mandelate Racemase

“…To effect a rate enhancement ( k cat / k non ) exceeding 15 orders of magnitude, MR stabilizes the altered substrate in the transition state (TS) by ∼26 kcal/mol. , This remarkable proficiency corresponds to an upper limit for the virtual dissociation constant for the complex of the enzyme and altered substrate in the TS (i.e., K tx ) equal to 2 × 10 –19 M, and therefore, MR is expected to be extremely sensitive to inhibition by analogues of either the TS or high-energy intermediate formed during catalysis . Consequently, we have been interested in developing TS analogue inhibitors of MR to ascertain whether there is a limit to the extent that the free energy of TS stabilization can be captured by an intermediate/TS analogue to effect strong binding . While analogues of the aci -carboxylate intermediate such as ( R , S )-α-hydroxybenzylphosphonate ( K i = 8.7 μM), benzohydroxamate (BzH, K i = 9.3 μM), Cupferron ( K i = 2.67 μM), and N -hydroxyformanilide ( K i = 2.79 μM), afforded binding affinities on the order of 100–375-fold greater than that of the substrate ( K m ≈ 1.0 mM), we recently discovered that phenylboronic acid (PBA, K i = 1.8 μM) and 4-chloro-PBA ( K i = 81 nM) were very potent inhibitors of MR .…”