The Role of Lysine 166 in the Mechanism of Mandelate Racemase from Pseudomonas putida: Mechanistic and Crystallographic Evidence for Stereospecific Alkylation by (R)-α-Phenylglycidate

The mechanism of irreversible inactivation of mandelate racemase (MR) from Pseudomonas putida by α-phenylgly cidate (αPGA) has been investigated stereochemically and crystallographically. The (R) and (S) enantiomers of αPGA were synthesized in high enantiomeric excess (81% ee and 83% ee, respectively) using Sharpless epoxidation chemistry. (R)-αPGA was determined to be a stereospecific and stoichiometric irreversible inactivator of MR. (S)-αPGA does not inactivate MR and appears to bind noncovalently to the active site of MR with less affinity than that of (R)-αPGA. The X-ray crystal structure (2.0-Å resolution) of MR inactivated by (R)-αPGA revealed the presence of a covalent adduct formed by nucleophilic attack of the ϵ-amino group of Lys 166 on the distal carbon of the epoxide ring of (R)-αPGA. The proximity of the α-proton of (S)-mandelate to Lys 166 [configurationally equivalent to (R)-αPGA] was corroborated by the crystal structure (2.1-Å resolution) of MR complexed with the substrate analog/ competitive inhibitor, (S)-atrolactate [(S)-α-methylmandelate]. These results support the proposal that Lys 166 is the polyvalent acid/base responsible for proton transfers on the (S) face of mandelate. In addition, the high-resolution structures also provide insight into the probable interactions of mandelate with the essential Mg²⁺ and functional groups in the active site.