The role of conformational flexibility of enzymes in the discrimination between amino acid and ester substrates for the subtilisin-catalyzed reaction in organic solvents.
To investigate how the conformational flexibility of subtilisin affects its ability to discriminate between enantiomeric amino acid and ester substrates for the subtilisin-catalyzed reaction in an organic solvent, the flexibility around the active site and the surface of subtilisin was estimated from the mobility of a spin label bound to subtilisin by ESR spectroscopy. Many studies on enzyme flexibility focus on the active site. Both the surface and active site flexibility play an important role in the enantioselectivity enhancement of the enzyme-catalyzed reaction. It was found, however, that the different behavior observed for the enantioselectivity between the amino acid and ester substrates could be correlated with the flexibility around the surface rather than the flexibility at the active site of subtilisin. In other words, for the ester substrates, the greater flexibility around the surface of subtilisin induced by a conformational change resulting from the presence of an additive such as DMSO is essential for the enantioselectivity enhancement. This model is also supported by the Michaelis-Menten kinetic parameters for each enantiomeric substrate. Our findings provide insight into the enantioselectivity enhancement for the resolution of enantiomers for enzyme-catalyzed reactions in organic solvents.
Watanabe
Keiichi
K
Graduate School of Science and Technology, Kobe University, Nada 657-8501, Japan.
Yoshida
Takashi
T
Ueji
Shin-ichi
S
D000596:Amino Acids / Q000737:chemistry*; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D004952:Esters / Q000737:chemistry*; D007700:Kinetics; D009795:Octanes / Q000737:chemistry*; D011487:Protein Conformation; D012997:Solvents / Q000737:chemistry; D013237:Stereoisomerism; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry*
D000596:Amino Acids; D004952:Esters; D009795:Octanes; D012997:Solvents; D013381:Subtilisins; C045798:2,2,4-trimethylpentane