spacer ON 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
Primary citation
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.
Authors Landro, J.A.search; Gerlt, J.A.search; Kozarich, J.W.search; Koo, C.W.search; Shah, V.J.search; Kenyon, G.L.search; Neidhart, D.J.search; Fujita, S.search; Petsko, G.A.search
Journal BIOCHEMISTRYsearch vol:33, pag:635-643 (1994), Identifiers: PubMed ID (8292591)search DOI (10.1021/bi00169a003)
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.
MeSH terms Alkylationsearch, Crystallographysearch, X-Raysearch, Epoxy Compoundssearch, Hydrogen-Ion Concentrationsearch, Ligandssearch, Lysinesearch, Modelssearch, Molecularsearch, Phenylpropionatessearch, Protein Conformationsearch, Pseudomonas putidasearch, Racemases and Epimerasessearch, Stereoisomerismsearch
Other entries described in this publication 1mdr
Secondary citations
Title Mechanism of the Reaction Catalyzed by Mandelate Racemase 2. Crystal Structure of Mandelate Racemase at 2.5 Angstroms Resolution: Identification of the Active Site and Possible Catalytic Residues
Authors Neidhart, D.J.search; Howell, P.L.search; Petsko, G.A.search; Powers, V.M.search; Li, R.search; Kenyon, G.L.search; Gerlt, J.A.search
Journal BIOCHEMISTRYsearch vol:30, pag:9264 (1991)
Title Mandelate Racemase and Muconate Lactonizing Enzyme are Mechanistically Distinct and Structurally Homologous
Authors Neidhart, D.J.search; Kenyon, G.L.search; Gerlt, J.A.search; Petsko, G.A.search
Journal NATUREsearch vol:347, pag:692 (1990)
Title Refined High-Resolution X-Ray Structures of Mandelate Racemase in Complex with Catalytically Active Metal Ions
Authors Neidhart, D.J.search
Journal To be Published
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