Citrate-condensing Enzyme-Oxalacetate Binary Complex: STUDIES ON ITS PHYSICAL AND CHEMICAL PROPERTIES

Abstract

Abstract A binary complex is formed between citrate-condensing enzyme and oxalacetate. Complex formation is accompanied by a change in protein conformation, as judged by changes in ultraviolet absorption, a small change in optical rotatory dispersion, and increased stability to denaturation. The stability of the complex in urea has made it possible to estimate that there is 1 oxalacetate molecule per active site on the enzyme, and that the dissociation constant of this complex is about 6 x 10-7 m. Urea treatment of the enzyme results in the exposure of sulfhydryl groups, changes in the ultraviolet absorption, changes in the optical rotatary dispersion spectrum indicative of unfolding, and changes in the sedimentation behavior. Urea treatment of the complex, however, shows essentially none of these changes. A number of analogues were tested as to their ability to bind to the oxalacetate site. On the basis of these studies it was concluded that oxalacetate was bound through both carboxyl groups, as well as through the carbonyl oxygen. It seems that once the oxalacetate site on the enzyme is occupied, then urea (or other denaturants) cannot react with the enzyme.