Trapping choline oxidase in a nonfunctional conformation by freezing at low pH.
Choline oxidase is a flavin-dependent enzyme that catalyzes the oxidation of choline to glycine-betaine, with oxygen as electron acceptor. Storage at pH 6 and -20 degrees C resulted in a change in the conformation of choline oxidase, which was associated with complete loss of catalytic activity when the enzyme was assayed at pH 6. Incubation of the inactive enzyme at pH values > or = 6.5 and 25 degrees C resulted in a fast and partial reactivation of the enzyme, which occurred with slow onset of steady state during enzymatic turnover. The rate of approaching steady state was independent of the concentrations of choline and enzyme, but increased to a limiting value with increasing pH, defining a pKa value of approximately 7.3 for an unprotonated group required for enzyme activation. Prolonged incubation of the inactive enzyme at pH 6 and temperatures > or = 20 degrees C, at which no hysteretic behavior was observed, resulted in the slow and full recovery of activity over 3 h, associated with a conformational change that reverted the enzyme to the native form. Activation of the enzyme at pH 6 was enthalpy-driven with deltaH(double dagger) and TdeltaS(double dagger) values of approximately 112 kJ mol(-1) and approximately 20 kJ mol(-1) determined at 25 degrees C. These data suggest that freezing the enzyme at low pH induces a localized and reversible conformational change that is associated with the complete and reversible loss of catalytic activity.
Hoang
Jane V
JV
Department of Chemistry, Georgia State University, Atlanta, Georgia 30302-4098, USA.
Gadda
Giovanni
G
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D001622:Betaine / Q000378:metabolism; D002794:Choline / Q000378:metabolism; D002942:Circular Dichroism; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D001622:Betaine; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D002794:Choline