Potent Inhibition of Mandelate Racemase by a Fluorinated Substrate-Product Analogue with a Novel Binding Mode

Abstract

Mandelate racemase (MR) from Pseudomonas putida catalyzes the Mg(2+)-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Because trifluorolactate is also a substrate of MR, we anticipated that replacing the phenyl rings of the competitive, substrate-product analogue inhibitor benzilate (Ki = 0.7 mM) with trifluoromethyl groups might furnish an inhibitor. Surprisingly, the substrate-product analogue 3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)propanoate (TFHTP) was a potent competitive inhibitor [Ki = 27 ± 4 μM; cf. Km = 1.2 mM for both (R)-mandelate and (R)-trifluorolactate]. To understand the origins of this high binding affinity, we determined the X-ray crystal structure of the MR-TFHTP complex to 1.68 Å resolution. Rather than chelating the active site Mg(2+) with its glycolate moiety, like other ground state analogues, TFHTP exhibited a novel binding mode with the two trifluoromethyl groups closely packed against the 20s loop and the carboxylate bridging the two active site Brønsted acid-base catalysts Lys 166 and His 297. Recognizing that positioning a carboxylate between the Brønsted acid-base catalysts could yield an inhibitor, we showed that tartronate was a competitive inhibitor of MR (Ki = 1.8 ± 0.1 mM). The X-ray crystal structure of the MR-tartronate complex (1.80 Å resolution) revealed that the glycolate moiety of tartronate chelated the Mg(2+) and that the carboxylate bridged Lys 166 and His 297. Models of tartronate in monomers A and B of the crystal structure mimicked the binding orientations of (S)-mandelate and that anticipated for (R)-mandelate, respectively. For the latter monomer, the 20s loop appeared to be disordered, as it also did in the X-ray structure of the MR triple mutant (C92S/C264S/K166C) complexed with benzilate, which was determined to 1.89 Å resolution. These observations indicate that the 20s loop likely undergoes a significant conformational change upon binding (R)-mandelate. In general, our observations suggest that inhibitors of other enolase superfamily enzymes may be designed to capitalize on the recognition of the active site Brønsted acid-base catalysts as binding determinants.

Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

“…(P) Twoc onformations of tartronate as observed bounda ttwo adjacent active siteso fMRf rom P. putida (PDB 4M6U) are shown. [94] Note that the comparison was created by superposingt wo subunits of the crystallographic dimer.The conformationc orresponding to the conformation observed for bound (S)-atrolactate (see panelO )isd esignated" ( S)"-tar,a nd the other conformation, presumably corresponding to ab ound (R)-substrate,i sd esignated "(R)"-tar. substrates in a mirror-image packing orientation as shown in Figure 2C.…”

Inactivation of Mandelate Racemase by 3-Hydroxypyruvate Reveals a Potential Mechanistic Link between Enzyme Superfamilies

“…± 0.3 mM ( Figure 1S of the Supporting Information)], binding with the same affinity as tartronate (Ki = 1.8 mM), 23 suggesting that the glycolate-like moiety present in both ligands chelates the Mg 2+ and the second carboxylate forms a salt bridge with the active site Brønsted acid-base catalysts, thereby compensating for the loss of hydrophobic interactions. 23 Pyruvate, which exists as the keto form under the assay conditions, 33,34 exhibited only 40% inhibition of MR activity at a concentration of 25 mM [i.e., Ki ≈ 20 mM, assuming competitive inhibition with 1 mM (R)-mandelate; Km = 1.2 mM].…”

“…23 Recognizing that these active site bases can be exploited as binding determinants for inhibitors, we showed that tartronate is also a competitive inhibitor of MR (Ki = 1.8 mM), binding with its glycolate moiety chelating the Mg 2+ ion and with the remaining carboxylate group interacting with and bridging the active site Brønsted acid-base catalysts. 23 Our interest in understanding the role of the active site Brønsted acid-base catalysts as binding determinants led us to interrogate the active site using three-carbon α-keto acids as tartronate analogues. Most unexpectedly, we discovered that 3-hydroxypyruvate (3-HP) is an irreversible inhibitor of MR, reacting at the active site to form a Schiff-base intermediate that is subsequently deprotonated to form an aldehyde/enol(ate) adduct.…”

“…23 Pyruvate, which exists as the keto form under the assay conditions, 33,34 exhibited only 40% inhibition of MR activity at a concentration of 25 mM [i.e., Ki ≈ 20 mM, assuming competitive inhibition with 1 mM (R)-mandelate; Km = 1.2 mM]. This low binding affinity is similar to that of lactate (Ki ≈ 30 mM) 20 and likely arises because the methyl group is not sufficiently hydrophobic, nor can it form salt bridges with the Brønsted acid-base catalysts.…”

“…23 Aside from 3-HP, all other ligands are not shown for the sake of clarity. The 3-HP-MR structure exhibits an "open" conformation of the 20s loop lid, similar to the conformation observed when the bulky substrate-product analogue benzilate is bound to C92S/C264S/K166C MR. 23 …”