The excluding effects of sucrose on a protein chemical degradation pathway: methionine oxidation in subtilisin.
The conformational stabilization of proteins by sucrose has been previously attributed to a preferential exclusion mechanism. The present study links this mechanism to stability against a chemical degradation pathway for subtilisin. Oxidation of a methionine residue adjacent to the active site to the sulfoxide form compromises subtilisin's enzymatic activity. In the presence of hydrogen peroxide and borate buffer, a borate-hydrogen peroxide complex binds to subtilisin's active site prior to the formation of methionine sulfoxide. Sucrose decreases the oxidation rate by limiting the accessibility of the complex to the methionine at the partially buried active site. The stabilization mechanism of sucrose is based on shifting the equilibrium of transiently expanding native conformations of subtilisin to favor the most compact states. Enzymatic parameter determination (kcat, KM) and hydrogen-deuterium exchange measurements confirm the limited conformational mobility of the enzyme in the presence of sucrose. Further support for limited mobility as the cause of oxidation inhibition by sucrose comes from the findings that neither viscosity nor possible interactions of sucrose with hydrogen peroxide, hydroxyl radicals, or borate can adequately explain the inhibition. The volume exclusion of sucrose from subtilisin is used to estimate the extent by which the native state of subtilisin must expand in solution to allow oxidation. The surface area of the oxidation-competent state is ca. 3.9% greater than that of the native state.
DePaz
R A
RA
Department of Chemical Engineering, University of Colorado, Boulder 80309, USA.
Barnett
C C
CC
Dale
D A
DA
Carpenter
J F
JF
Gaertner
A L
AL
Randolph
T W
TW
D001881:Borates / Q000378:metabolism; D003903:Deuterium / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D016166:Free Radical Scavengers / Q000494:pharmacology; D006859:Hydrogen / Q000378:metabolism; D008715:Methionine / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D020860:Subtilisin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D013395:Sucrose / Q000494:pharmacology*; D014783:Viscosity / Q000187:drug effects
D001881:Borates; D016166:Free Radical Scavengers; D013395:Sucrose; D006859:Hydrogen; D008715:Methionine; D003903:Deuterium; D020860:Subtilisin