Glutamate Racemase Dimerization Inhibits Dynamic Conformational Flexibility and Reduces Catalytic Rates

• Published in: Biochemistry (Easton) Vol. 48; no. 29; pp. 7045 - 7055
• Main Authors: Mehboob, Shahila, Guo, Liang, Fu, Wentao, Mittal, Anuradha, Yau, Tiffany, Truong, Kent, Johlfs, Mary, Long, Fei, Fung, Leslie W.-M, Johnson, Michael E
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 28.07.2009
• Subjects: Amino Acid Isomerases - chemistry; Amino Acid Isomerases - genetics; Amino Acid Isomerases - metabolism; BASIC BIOLOGICAL SCIENCES; Biocatalysis; CATALYSIS; Chromatography, Gel; DIMERIZATION; DIMERS; ENZYMES; FLEXIBILITY; GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; MUTANTS; MUTATIONS; NORMAL-MODE ANALYSIS; Nuclear Magnetic Resonance, Biomolecular; PRECURSOR; Protein Conformation; RESIDUES; SCATTERING; Scattering, Radiation; SYNTHESIS
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9005072

Glutamate racemase (RacE) is a bacterial enzyme that converts l-glutamate to d-glutamate, an essential precursor for peptidoglycan synthesis. In prior work, we have shown that both isoforms cocrystallize with d-glutamate as dimers, and the enzyme is in a closed conformation with limited access to the active site [May, M., et al. (2007) J. Mol. Biol. 371, 1219−1237]. The active site of RacE2 is especially restricted. We utilize several computational and experimental approaches to understand the overall conformational dynamics involved during catalysis when the ligand enters and the product exits the active site. Our steered molecular dynamics simulations and normal-mode analysis results indicate that the monomeric form of the enzyme is more flexible than the native dimeric form. These results suggest that the monomeric enzyme might be more active than the dimeric form. We thus generated site-specific mutations that disrupt dimerization and find that the mutants exhibit significantly higher catalytic rates in the d-Glu to l-Glu reaction direction than the native enzyme. Low-resolution models restored from solution X-ray scattering studies correlate well with the first six normal modes of the dimeric form of the enzyme, obtained from NMA. Thus, along with the local active site residues, global domain motions appear to be implicated in the catalytically relevant structural dynamics of this enzyme and suggest that increased flexibility may accelerate catalysis. This is a novel observation that residues distant from the catalytic site restrain catalytic activity through formation of the dimer structure.