1DTN

MANDELATE RACEMASE MUTANT D270N CO-CRYSTALLIZED WITH (S)-ATROLACTATE

Summary for 1DTN

• Entry DOI: 10.2210/pdb1dtn/pdb
• Descriptor: MANDELATE RACEMASE, MAGNESIUM ION, ATROLACTIC ACID (2-PHENYL-LACTIC ACID), ... (4 entities in total)
• Functional Keywords: racemase, isomerase, mandelate pathway
• Biological source: Pseudomonas aeruginosa
• Total number of polymer chains: 1
• Total formula weight: 38792.09

Authors

Clifton, J.G.,Petsko, G.A. (deposition date: 1996-03-28, release date: 1996-10-14, Last modification date: 2024-02-07)

Primary citation

Mitra, B.,Kallarakal, A.T.,Kozarich, J.W.,Gerlt, J.A.,Clifton, J.G.,Petsko, G.A.,Kenyon, G.L.
Mechanism of the reaction catalyzed by mandelate racemase: importance of electrophilic catalysis by glutamic acid 317.
Biochemistry, 34:2777-2787, 1995

PubMed Abstract: In the high-resolution X-ray structure of mandelate racemase (MR) with the competitive inhibitor (S)-atrolactate bound in the active site [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., & Petsko, G. A. (1994) Biochemistry 33, 635-643], the carboxylic acid group of Glu 317 is hydrogen-bonded to the carboxylate group of the bound inhibitor. This geometry suggests that the carboxylic acid functional group of Glu 317 participates as a general acid catalyst in the concerted general acid-general base catalyzed formation of a stabilized enolic tautomer of mandelic acid as a reaction intermediate. To test this hypothesis, the E317Q mutant of MR was constructed and subjected to high-resolution X-ray structural analysis in the presence of (S)-atrolactate. No conformational alterations were observed to accompany the E317Q substitution at 2.1 A resolution. The values for kcat were reduced 4.5 x 10(3)-fold for (R)-mandelate and 2.9 x 10(4)-fold for (S)-mandelate; the values for kcat/Km were reduced 3 x 10(4)-fold. The substrate and solvent deuterium isotope effects measured for both wild-type MR and the E317Q mutant are not multiplicative when deuteriated substrate is studied in D2O, which suggests that the reactions catalyzed by both enzymes are stepwise and involve the formation of stabilized enolic intermediates. In contrast to wild-type MR, E317Q does not catalyze detectable elimination of bromide ion from either enantiomer of p-(bromomethyl)mandelate. However, E317Q is irreversibly inactivated by racemic alpha-phenylglycidate at a rate comparable to that measured for wild-type MR. Taken together, these mechanistic properties confirm the importance of Glu 317 as a general acid catalyst in the reaction catalyzed by wild-type MR. The kcat for wild-type MR and the reduction in kcat observed for E317O are discussed in terms of the analysis recently described by Gerlt and Gassman for understanding the rates and mechanisms of enzyme-catalyzed proton abstraction reactions from carbon acids [Gerlt, J. A., & Gassman, P. G. (1993) J. Am. Chem. Soc. 115, 11552-11568; Gerlt, J. A., & Gassman, P. G. (1993) Biochemistry 32, 11943-11952].

PubMed: 7893689
DOI: 10.1021/bi00009a006

Experimental method

X-RAY DIFFRACTION (2.1 Å)