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Transition state stabilization of subtilisins in organic media (1994)

Xu, Zu-Feng ; Affleck, Rhett ; Wangikar, Pramod ; [et al.]

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 43 (1994), S. 515-520

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ISSN: 0006-3592

Keywords: subtilisin ; organic solvents ; transition state stabilization ; EPR spectroscopy ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Electrostatic forces are among the stabilizing interactions that contribute to the high degree of enzyme-transition state complementarity. The active-site polarity, which can differ substaintially from that of water, is thus an important determinant of transition state stabilization. Here we pose the question of whether the rate of an enzymatic reaction proceeding through a charged transition state can be increased by increasing the active-site polarity in an organic solvent. The active-site polarity of subtilisin has been reduced by dehydration and suspension in a nonpolar solvent (tetrahydrofuran), and then increased by adding water to the solvent. Enhancing the local polarity substantially increasing the rate of catalysis, implicating polarity as an important factor in stabilizing the charged tetrahedral transition state. Studies with subtilisins whose active sites have been modified by site-directed mutagenesis support the role of polarity in transition state stabilization. © 1994 John Wiley & Sons, Inc.

Additional Material: 3 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/bit.260430612

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High pressure EPR studies of protein mobility in reversed micelles (1994)

Affleck, Rhett ; Clark, Douglas S. ; Kamat, Sanjay ; [et al.]

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 43 (1994), S. 342-348

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ISSN: 0006-3592

Keywords: organic solvents ; reversed micelles ; protein mobility ; EPR spectra of enzymes ; Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: We have investigated the effect of pressure on structural properties of subtilisin solubilized in reversed micelles of Tween-85/isopropanol in hexane. Electron paramagnetic resonance (EPR) spectra of spin-labeled enzyme indicate a reduction in spin-label mobility when the enzyme is transferred from aqueous solution to the microemulsion. One explanation for the spectral broadening is a change in the protein's active-site conformation and/or dynamics. However, over a W0 range of 80 to 180, EPR spectroscopy could detect no change in the enzyme's environment, conformation, or molecular dynamics. The EPR spectra also contained a contribution from free spin label located in an environment with a polarity roughly between that of propanol and bulk water. No changes in the polarity surrounding the free spin label nor in the enzyme's structural properties were evident at pressures up to 10,000 psi. Previous work has demonstrated that pressure can be used to manipulate the size of some reversed micelles, and the EPR data indicated that for this system such pressure tuning of micellar properties will not adversely affect the structure of solubilized enzyme. © 1994 John Wiley & Sons, Inc.

Additional Material: 5 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/bit.260430412

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