Conformational Changes at the Active Site of D-Glyceraldehyde-3-Phosphate Dehydrogenase During Unfolding in Guanidine Solution

Abstract

The fluorescent NAD derivative, covalently linked to the active site sulfhydryl groups of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase (Tsou, C.L. et al., Biochem. Soc. Trans. 11, 425–429, 1983), has been used as a probe to study the conformational change at the active site of this enzyme during unfolding by guanidine denaturation. This derivative shows both a red shift of its emission maximum and a decrease in fluorescence intensity at the same guanidine concentration which brought about complete inactivation together with similar changes of the intrinsic protein fluorescence. Complete unfolding of the enzyme as indicated by further red shift in the emission maximum and decrease in intensity of the intrinsic fluorescence requires much higher guanidine concentrations. It appears that the low guanidine concentrations required to bring about complete inactivation also lead to perturbation of the active site conformation and that a Trp residue is situated at or near the active site region.