Effect of modification of SH-groups in D-glyceraldehyde-3-phosphate dehydrogenase on the properties of enzyme--coenzyme complex.

The NAD-binding of pig muscle glyceraldehyde-3-phosphate dehydrogenase after modifying certain SH-groups of the enzyme was studied by spectrophotometric titration and gel-chromatography. The enzyme modified on residue Cys-149 with N-ethylmaleimide, iodoacetate or p-mercuribenzoate binds NAD less tightly than does the unmodified enzyme. However, the differences between the NAD-binding sites characteristic of the native tetrameric enzyme are largely retained in the modified enzyme. Residue Cys-153 is near to the surface of the enzyme and is temporarily exposed due to the fluctuation of the protein. It can be modified only after blocking Cys-149. Modification of residue Cys-153 with N-ethylmaleimide does not further influence NAD-binding. Reaction of Cys-153 with p-mercuribenzoate does not directly cause the release of NAD. In this case the enzyme-coenzyme complex decomposes due to secondary conformational changes. This followed from the finding that the disappearance of the absorption band characteristic of the enzyme-coenzyme charge transfer complex is a first order process is equal to that of the structural changes following mercaptide formation of Cys-153.