CIRCULAR PERMUTATION WITHIN THE COENZYME BINDING DOMAIN OF THE TETRAMERIC GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM BACILLUS-STEAROTHERMOPHILUS

A circularly permuted (cp) variant of the phosphorylating NAD-dependen t glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stear othermophilius has been constructed with N- and C-termini created with in the coenzyme binding domain. The cp variant has a k(cat) value equa l to 40% of the wild-type value, whereas K-m and KD values for NAD sho w a threefold decrease compared to wild type. These results indicate t hat the folding process and the conformational changes that accompany NAD binding during the catalytic event occur efficiently in the permut ed variant and that NAD binding is tighter. Reversible denaturation ex periments show that the stability of the variant is only reduced by 0. 7 kcal/mol compared to the wild-type enzyme. These experiments confirm and extend results obtained recently on other permuted proteins. For multimeric proteins, such as GAPDH, which harbor subunits with two str uctural domains, the natural location of the N- and C-termini is not a prerequisite for optimal folding and biological activity.