ATP BINDING AND HYDROLYSIS BY THE MULTIFUNCTIONAL PROTEIN DISULFIDE-ISOMERASE

We previously reported the ability of protein disulfide isomerase (PDI ) to undergo an ATP-dependent autophosphorylation. Our efforts to map the modification site have been hindered by the low abundance and inst ability of the labeling. Results are presented in this paper on the na ture of phospho-PDI, which appears as an intermediate with a half-life of 2.5-8.8 min in an ATPase reaction. ATP binds to PDI with high affi nity, K-d 9.66 mu M, and the kinetic parameters K-m ATP and k(cat) of the ATPase reaction were measured by using a pyruvate kinase-lactate d ehydrogenase-coupled assay under various conditions. Strikingly, the A TPase reaction is stimulated in the presence of denatured polypeptides , while the disulfide oxidization activity of PDI is not affected by A TP. However, PDI is known to participate in various unrelated function s in the endoplasmic reticulum, and ATP could be involved in the regul ation of one of these. The results are discussed in light of recent fi ndings on ATP-chaperone relationships.