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

Area of Science:

• Enzymology
• Biochemistry
• Chemical Biology

Background:

• Mandelate racemase (MR) is a key enzyme in mandelate metabolism, catalyzing the interconversion of (R)- and (S)-mandelate.
• MR stabilizes the transition state (TS) of this reaction by approximately 26 kcal/mol.
• Enzymes serve as models to study how transition state (TS) analogs can achieve strong binding.

Purpose of the Study:

• To investigate the binding thermodynamics of halogen-substituted phenylboronic acids (PBAs) to MR.
• To identify potent inhibitors of MR by exploring the impact of halogen substitution on binding affinity.
• To understand the molecular interactions driving the binding of PBAs to MR.

Main Methods:

• Determination of thermodynamic parameters for PBA binding to MR.
• Enzyme inhibition assays to determine binding constants (Kdapp, Ki).
• pH-dependence studies and 11B NMR spectroscopy to characterize inhibitor binding modes.
• Linear free energy relationship analysis to compare inhibitor binding with enzyme catalysis.

Main Results:

• Binding of PBAs to MR was primarily driven by favorable entropy changes.
• 3,4-Dichloro-PBA emerged as the most potent MR inhibitor identified, with a Kdapp of 11 ± 2 nM, binding ~72,000-fold stronger than the substrate.
• MR preferentially binds the anionic, tetrahedral form of 3,4-dichloro-PBA, with a pH-independent Ki of 5.7 ± 0.5 nM.
• Analysis indicated that MR recognizes 3,4-dichloro-PBA as a transition state (TS) analog.

Conclusions:

• Halogen substitution on PBAs can significantly enhance binding affinity to MR by exploiting dispersion forces and capturing additional transition state (TS) stabilization energy.
• The study provides a framework for designing potent enzyme inhibitors by optimizing interactions with the enzyme's transition state (TS).
• Understanding these interactions is crucial for developing targeted therapeutics and biochemical tools.