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 instability of the labeling. Results are presented in this paper on the nature 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 affinity, Kd 9.66 microM, and the kinetic parameters KmATP and kcat of the ATPase reaction were measured by using a pyruvate kinase-lactate dehydrogenase-coupled assay under various conditions. Strikingly, the ATPase reaction is stimulated in the presence of denatured polypeptides, while the disulfide oxidization activity of PDI is not affected by ATP. However, PDI is known to participate in various unrelated functions in the endoplasmic reticulum, and ATP could be involved in the regulation of one of these. The results are discussed in light of recent findings on ATP-chaperone relationships.