ORGN 701 |
| Erika A. Taylor Ringia, Department of Chemistry, University of Illinois, 600 S. Mathews Ave., 102-5, Urbana, IL 61801, James B. Thoden, Department of Biochemistry, University of Wisconsin - Madison, 433 Babcock Drive, Room 2224B, Madison, WI 53706, and John A. Gerlt, Department of Biochemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801. |
| o-Succinylbenzoate synthase (OSBS) from Amycolaptosis catalyzes an exergonic dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1S-carboxylate (SHCHC) to 4-(2’-carboxylphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB) with a kcat/KM of 2.5 + 0.6 x 105 M-1 s-1. This enzyme was originally identified as “N-acylamino acid racemase” (NAAAR) with a reported kcat/KM for the best substrate, N-acetyl-L-methionine (NALM), of 3.1 x 102 M-1 s-1. The rate of this adventitious racemization was optimized using compounds that mimic SHCHC to give rate constants approaching the dehydration. Mutants of Lys residues 163 and 263 were constructed to explore both the NAAAR and OSBS reactions. These mutants displayed no activity for either reaction, however stereospecific exchange of the a-proton of R- and S- acylamino acids was observed for the Lys 163 and Lys 263 mutants, respectively, supporting the prediction of an enediolate anion intermediate. Mutational and crystallographic evidence support a one-base mechanism for the OSBS reaction and a two-base mechanism for the NAAAR reaction. |
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Proteins, Peptides, Amino Acids, and Nucleotides
8:30 AM-11:30 AM, Thursday, September 11, 2003 Sheraton New York -- Royal Ballroom B, Oral
Division of Organic Chemistry |