Value of a Hydrogen Bond in Triosephosphate Isomerase Loop Motion

• Published in: Biochemistry (Easton) 2007-05, Vol.46 (20), p.6001-6010
• Main Authors: Berlow, Rebecca B, Igumenova, Tatyana I, Loria, J. Patrick
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites - genetics; Catalysis; Chickens; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Phenylalanine - genetics; Protein Conformation; Protein Structure, Tertiary - genetics; Thermodynamics; Triose-Phosphate Isomerase - chemistry; Triose-Phosphate Isomerase - genetics; Tyrosine - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi700344v

The motion of the active site loop (loop 6) in triosephosphate isomerase is investigated in solution by TROSY NMR spin-relaxation experiments. The data show clear evidence for motion with an exchange rate constant (k ex) of 9000 s-1, consistent with opening and closing of this loop being partially rate-limiting to catalytic throughput. Similar rate constants are observed for residues in both the N- and C-terminal regions of loop 6, suggesting motional coupling of the loop hinges. Mutation of tyrosine 208 to a phenylalanine (Y208F) eliminates a hydrogen bond in the closed loop conformation. NMR experiments with this mutant enzyme indicate an increase in the population of the open conformer and concomitant increase in the opening rate constant and a decrease in the rate of loop closure. The destabilization of the closed conformer by ∼3 kJ/mol is consistent with a similar decrease in affinity of Y208F for ligand. The site-specific nature of these experiments leads to additional insight into loop 6 motion and the role of a conserved residue in modulating this motion.