title: The role of lysine 166 in the mechanism of mandelate racemase from Pseudomonas putida: mechanistic and crystallographic evidence for stereospecific alkylation by (R)-alpha-phenylglycidate.
abstract: The mechanism of irreversible inactivation of mandelate racemase (MR) from Pseudomonas putida by alpha-phenylglycidate (alpha PGA) has been investigated stereochemically and crystallographically. The (R) and (S) enantiomers of alpha PGA were synthesized in high enantiomeric excess (81% ee and 83% ee, respectively) using Sharpless epoxidation chemistry. (R)-alpha PGA was determined to be a stereospecific and stoichiometric irreversible inactivator of MR. (S)-alpha PGA does not inactivate MR and appears to bind noncovalently to the active site of MR with less affinity than that of (R)-alpha PGA. The X-ray crystal structure (2.0-A resolution) of MR inactivated by (R)-alpha PGA revealed the presence of a covalent adduct formed by nucleophilic attack of the epsilon-amino group of Lys 166 on the distal carbon on the epoxide ring of (R)-alpha PGA. The proximity of the alpha-proton of (S)-mandelate to Lys 166 [configurationally equivalent to (R)-alpha PGA] was corroborated by the crystal structure (2.1-A resolution) of MR complexed with the substrate analog/competitive inhibitor, (S)-atrolactate [(S)-alpha-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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
authors.forename: J A
authors.initials: JA
authors.lastname: Landro
authors.forename: J A
authors.initials: JA
authors.lastname: Gerlt
authors.forename: J W
authors.initials: JW
authors.lastname: Kozarich
authors.forename: C W
authors.initials: CW
authors.lastname: Koo
authors.forename: V J
authors.initials: VJ
authors.lastname: Shah
authors.forename: G L
authors.initials: GL
authors.lastname: Kenyon
authors.forename: D J
authors.initials: DJ
authors.lastname: Neidhart
authors.forename: S
authors.initials: S
authors.lastname: Fujita
authors.forename: G A
authors.initials: GA
authors.lastname: Petsko
mesh_terms: D000478:Alkylation; D018360:Crystallography, X-Ray; D004852:Epoxy Compounds / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008239:Lysine; D008958:Models, Molecular; D010666:Phenylpropionates / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000201:enzymology*; D019998:Racemases and Epimerases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D004852:Epoxy Compounds; D008024:Ligands; D010666:Phenylpropionates; C008851:alpha-phenylglycidate; D019998:Racemases and Epimerases; C021540:mandelate racemase; D008239:Lysine