Interrogating l-fuconate dehydratase with tartronate and 3-hydroxypyruvate reveals subtle differences within the mandelate racemase-subgroup of the enolase superfamily.

Enzymes of the enolase superfamily share a conserved structure and a common partial reaction (i.e., metal-assisted, Brønsted base-catalyzed enol(ate) formation). The architectures of the enolization apparatus at the active sites of the mandelate racemase (MR)-subgroup members MR and l-fuconate dehydratase (FucD) are almost indistinguishable at the structural level. Tartronate and 3-hydroxypyruvate (3-HP) recognize the enolization apparatus and can be used to interrogate the active sites for differences that may not be apparent from structural data. We report a circular dichroism-based assay of FucD activity that monitors the change in ellipticity at 216 nm (Δ[Θ]S-P = 8985 ± 87 deg cm2 mol-1) accompanying the conversion of l-fuconate to 2-keto-3-deoxy-l-fuconate. Tartronate was a linear mixed-type inhibitor of FucD (Ki = 8.4 ± 0.7 mM, αKi = 63 ± 11 mM), binding 18-fold weaker than l-fuconate, compared with 2-fold weaker binding of tartronate by MR relative to mandelate. 3-HP irreversibly inactivated FucD (kinact/KI = 0.018 ± 0.002 M-1s-1) with an efficiency that was ∼4.6 × 103-fold less than that observed with MR. The inactivation arose predominantly from modifications at multiple sites and Tris-HCl, but not l-fuconate, afforded protection against inactivation. Similar to the reaction of 3-HP with MR, 3-HP modified the Brønsted base catalyst (Lys 220) at the active site of FucD, which was facilitated by the Brønsted acid catalyst His 351. Thus, the interactions of tartronate and 3-HP with MR and FucD revealed differences in binding affinity and reactivity that differentiated between the enzymes' enolization apparatuses.

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

Mandelate racemase (MR) catalyzes the Mg2+-dependent interconversion of (R)- and (S)-mandelate by stabilizing the altered substrate in the transition state (TS) by ∼26 kcal/mol. The enzyme has been employed as a model to explore the limits to which the free energy of TS stabilization may be captured by TS analogues to effect strong binding. Herein, we determined the thermodynamic parameters accompanying binding of a series of bromo-, chloro-, and fluoro-substituted phenylboronic acids (PBAs) by MR and found that binding was predominately driven by favorable entropy changes. 3,4-Dichloro-PBA was discovered to be the most potent inhibitor yet identified for MR, binding with a Kdapp value of 11 ± 2 nM and exceeding the binding of the substrate by ∼72,000-fold. The ΔCp value accompanying binding (-488 ± 18 cal·mol-1 K-1) suggested that dispersion forces contribute significantly to the binding. The pH-dependence of the inhibition revealed that MR preferentially binds the anionic, tetrahedral form of 3,4-dichloro-PBA with a pH-independent Ki value of 5.7 ± 0.5 nM, which was consistent with the observed upfield shift of the 11B NMR signal. The linear free energy relationship between log(kcat/Km) and log(1/Ki) for wild-type and 11 MR variants binding 3,4-dichloro-PBA had a slope of 0.8 ± 0.2, indicating that MR recognizes the inhibitor as an analogue of the TS. Hence, halogen substitution may be utilized to capture additional free energy of TS stabilization arising from dispersion forces to enhance the binding of boronic acid inhibitors by MR.

Altering the binding determinant on the interdigitating loop of mandelate racemase shifts specificity towards that of d-tartrate dehydratase.

The enolase superfamily (ENS) has served as a paradigm for understanding how enzymes that share a conserved structure, as well as a common partial reaction (i.e., metal-assisted, Brønsted base-catalyzed enol(ate) formation), evolved from a common progenitor to catalyze mechanistically diverse reactions. Enzymes of the mandelate racemase (MR)-subgroup of the ENS share interdigitating loops between adjacent, 2-fold symmetry-related protomers of the tightly associated homodimers that comprise their quaternary structures. For the MR-subgroup members MR and d-tartrate dehydratase (TarD), the tip of the loop contributes a binding determinant to the adjacent active site (i.e., Leu 93 and Lys 102, respectively). To assess the role of Leu 93 of MR in substrate specificity and catalysis, we constructed L93 variants bearing hydrophobic (L93A, L93F, and L93W), polar neutral (L93N), acidic (L93D), or basic (L93K and L93R) residues at position 93. Gel filtration-HPLC revealed that wild-type MR and all L93 MR variants, apart from L93R MR (dimeric), were tetrameric in solution. The catalytic efficiency (kcat/Km) was reduced in the R→S and S→R reaction directions for all variants, primarily due to reduced turnover (kcat). Substitution of Leu 93 by Lys or Arg to mimic Lys 102 of TarD enhanced the binding of malate and tartrate, with meso- and d-tartrate exhibiting linear mixed-type inhibition of L93K MR. Despite the striking 500-fold increase in the binding affinity of d-tartrate, relative to (S)-mandelate, L93K MR exhibited no TarD activity. MD simulations suggested that the failure of L93K MR to catalyze α-deprotonation (i.e., H-D exchange) arises from inappropriate positioning of the Brønsted base (Lys 166). Thus, a change in binding determinant on the interdigitating loop can play a significant role in governing substrate specificity within the ENS, but does not necessarily confer 'new' catalytic activity despite similarities in catalytic machinery.

Slow-Onset, Potent Inhibition of Mandelate Racemase by 2-Formylphenylboronic Acid. An Unexpected Adduct Clasps the Catalytic Machinery.

o-Carbonyl arylboronic acids such as 2-formylphenylboronic acid (2-FPBA) are employed in biocompatible conjugation reactions with the resulting iminoboronate adduct stabilized by an intramolecular N-B interaction. However, few studies have utilized these reagents as active site-directed enzyme inhibitors. We show that 2-FPBA is a potent reversible, slow-onset inhibitor of mandelate racemase (MR), an enzyme that has served as a valuable paradigm for understanding enzyme-catalyzed abstraction of an α-proton from a carbon acid substrate with a high pKa. Kinetic analysis of the progress curves for the slow onset of inhibition of wild-type MR using a two-step kinetic mechanism gave Ki and Ki* values of 5.1 ± 1.8 and 0.26 ± 0.08 µM, respectively. Hence, wild-type MR binds 2-FPBA with an affinity that exceeds that for the substrate by ∼3000-fold. K164R MR was inhibited by 2-FPBA, while K166R MR was not inhibited, indicating that Lys 166 was essential for inhibition. Unexpectedly, mass spectrometric analysis of the NaCNBH3-treated enzyme-inhibitor complex did not yield evidence of an iminoboronate adduct. 11B nuclear magnetic resonance spectroscopy of the MR·2-FPBA complex indicated that the boron atom was sp3-hybridized (δ 6.0), consistent with dative bond formation. Surprisingly, X-ray crystallography revealed the formation of an Nζ-B dative bond between Lys 166 and 2-FPBA with intramolecular cyclization to form a benzoxaborole, rather than the expected iminoboronate. Thus, when o-carbonyl arylboronic acid reagents are employed to modify proteins, the structure of the resulting product depends on the protein architecture at the site of modification.