title: Structural and catalytic response to temperature and cosolvents of carboxylesterase EST1 from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1.
abstract: The interactive effects of temperature and cosolvents on the kinetic and structural features of a carboxylesterase from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1 (Sso EST1) were examined. While dimethylformamide, acetonitrile, and dioxane were all found to be deleterious to enzyme function, dimethyl sulfoxide (DMSO) activated Sso EST1 to various extents. This was particularly true at 3.5% (v/v) DMSO, where k(cat) was 20-30% higher than at 1.2% DMSO, over the temperature range of 50-85 degrees C. DMSO compensated for thermal activation in some cases; for example, k(cat) at 60 degrees C in 3.5% DMSO was comparable to k(cat) at 85 degrees C in 1.2% DMSO. The relationship between DMSO activation and enzyme structural characteristics was also investigated. Nuclear magnetic resonance spectroscopy and circular dichroism showed no gross change in enzyme conformation with 3.5% DMSO between 50 and 80 degrees C. However, low levels of DMSO were shown to have a small yet significant change in enzyme conformation. This was evident through the reduction of Sso EST1's melting temperature and changes in the microenvironment of the enzyme's tyrosine and tryptophan residues at 3.5% versus 1.2% (v/v) solvent. Finally, activation parameter analysis based on kinetic data, at 1.2% and 3.5% DMSO, implied an increase in conformational flexibility with additional cosolvent. These results suggest the activating effect of DMSO was related to small changes in the enzyme's structure resulting in an increase in its conformational flexibility. Thus, in addition to their use for solubilizing hydrophobic substrates in water, cosolvents may also serve as activators in applications involving thermostable biocatalysts at sub-optimal temperatures.
authors.affiliation: Department of Chemical Engineering, North Carolina State University, Raleigh 27695-7905, USA.
authors.forename: Amitabh C
authors.initials: AC
authors.lastname: Sehgal
authors.forename: Richele
authors.initials: R
authors.lastname: Tompson
authors.forename: John
authors.initials: J
authors.lastname: Cavanagh
authors.forename: Robert M
authors.initials: RM
authors.lastname: Kelly
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D002384:Catalysis; D004121:Dimethyl Sulfoxide / Q000737:chemistry*; D004126:Dimethylformamide / Q000737:chemistry; D004146:Dioxanes / Q000737:chemistry; D004789:Enzyme Activation; D004795:Enzyme Stability; D008968:Molecular Conformation; D011786:Quality Control; D012680:Sensitivity and Specificity; D012997:Solvents / Q000737:chemistry*; D013045:Species Specificity; D013329:Structure-Activity Relationship; D016944:Sulfolobus / Q000145:classification / Q000201:enzymology; D013696:Temperature
chemical_list: D000097:Acetonitriles; D004146:Dioxanes; D012997:Solvents; D004126:Dimethylformamide; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; C025223:1,4-dioxane; D004121:Dimethyl Sulfoxide; C032159:acetonitrile