Phosphate ion partially relieves the cooperativity of effector binding in D-3-phosphoglycerate dehydrogenase without altering the cooperativity of inhibition

The binding of L-serine to phosphoglycerate dehydrogenase from E. coli disp lays elements of both positive and negative cooperativity. In addition, the inhibition of enzymatic activity by L-serine is also cooperative with Hill coefficients greater than 1. However, phosphate buffer significantly reduc es the cooperative effects in serine binding without affecting the cooperat ivity of inhibition of activity. The maximal degree of inhibition and fluor escence quenching in Tris buffer occurs when an average of two serine bindi ng sites out of four are occupied. This value increases to three out of the four sites at maximal levels of inhibition and quenching in phosphate buff er. The increase from two to three sites appears to be due to the ability o f phosphate to reduce the site to site cooperative effects and render each ligand binding site less dependent on each other. The correlation between t he level of inhibition and the fractional site occupancy indicates that in Tris buffer, one serine is bound to each interface at maximal effect. In th e presence of phosphate, the order of binding appears to change so that bot h sites at one interface fill before the first site at the opposite interfa ce is occupied. In each case, there is a good correlation between serine bi nding, conformational change at the regulatory site interfaces, and inhibit ion of enzyme activity. The observation that phosphate does not appear to h ave a similar effect on the cooperativity of inhibition of enzymatic activi ty suggests that there are two distinct cooperative pathways at work: one p ath between the four serine binding sites, and one path between the serine binding sites and the active sites.