1dtn: Difference between revisions
No edit summary |
No edit summary |
||
| Line 5: | Line 5: | ||
==Overview== | ==Overview== | ||
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 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]. | ||
==About this Structure== | ==About this Structure== | ||
1DTN is a | 1DTN is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_aeruginosa Pseudomonas aeruginosa] with MG and APG as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Mandelate_racemase Mandelate racemase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=5.1.2.2 5.1.2.2] Structure known Active Sites: ACT, CAR and MTL. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1DTN OCA]. | ||
==Reference== | ==Reference== | ||
| Line 23: | Line 23: | ||
[[Category: racemase]] | [[Category: racemase]] | ||
''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 5 12:34:48 2007'' | ||
Revision as of 13:29, 5 November 2007
|
MANDELATE RACEMASE MUTANT D270N CO-CRYSTALLIZED WITH (S)-ATROLACTATE
OverviewOverview
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].
About this StructureAbout this Structure
1DTN is a Single protein structure of sequence from Pseudomonas aeruginosa with MG and APG as ligands. Active as Mandelate racemase, with EC number 5.1.2.2 Structure known Active Sites: ACT, CAR and MTL. Full crystallographic information is available from OCA.
ReferenceReference
Mechanism of the reaction catalyzed by mandelate racemase: importance of electrophilic catalysis by glutamic acid 317., Mitra B, Kallarakal AT, Kozarich JW, Gerlt JA, Clifton JG, Petsko GA, Kenyon GL, Biochemistry. 1995 Mar 7;34(9):2777-87. PMID:7893689
Page seeded by OCA on Mon Nov 5 12:34:48 2007