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

James A. Landro, John A. Gerlt, John W. Kozarich, Carolyn W. Koo, Vibhakar J. Shah, George L. Kenyon, David J. Neidhart, Shigeo Fujita, Gregory A. Petsko

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)635-643
Number of pages9
JournalBiochemistry
Volume33
Issue number3
DOIs
StatePublished - Jan 1 1994
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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