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Entropy effects on protein hinges: the reaction catalyzed by triosephosphate isomerase.

Biochemistry. 2004 Sep 14;43(36):11436-45. doi:10.1021/bi049208d
Jingyi Xiang 1 , Ju-yeon Jung , Nicole S Sampson
Jingyi Xiang 1 , Ju-yeon Jung , Nicole S Sampson

[No authors listed]

Author information
  • 1 Department of Chemistry, State University of New York, Stony Brook, New York 11794-3400, USA.

摘要


Many proteins utilize segmental motions to catalyze a specific reaction. The Omega loop of triosephosphate isomerase (TIM) is important for preventing the loss of the reactive enediol(ate) intermediate. The loop opens and closes even in the absence of the ligand, and the loop itself does not change conformation during movement. The conformational changes are localized to two hinges at the loop termini. Glycine is never observed in native TIM hinge sequences. In this paper, the hypothesis that limited access to conformational space is a requirement for protein hinges involved in catalysis was tested. The N-terminal hinge was mutated to P166/V167G/W168G (PGG), and the C-terminal hinge was mutated to K174G/T175G/A176G (GGG) in chicken TIM. The single-hinge mutants PGG and GGG had k(cat) values 200-fold lower than that of the wild type and K(m) values 10-fold higher. The k(cat) of double-hinge mutant P166/V167G/W168G/K174G/T175G/A176G was reduced 2500-fold; the K(m) was 10-fold higher. A combination of primary kinetic isotope effect measurements, isothermal calorimetric measurements, and (31)P NMR spectroscopic titration with the inhibitor 2-phosphoglycolate revealed that the mutants have a different ligand-binding mode than that of the wild-type enzyme. The predominant conformations of the mutants even in the presence of the inhibitor are loop-open conformations. In conclusion, mutation of the hinge residues to glycine resulted in the sampling of many more hinge conformations with the consequence that the population of the active-closed conformation is reduced. This reduced population results in a reduced catalytic activity.

基因