D-3-Phosphoglycerate dehydrogenase (PGDH) (EC 1.1.1.95) from Escherichia coli is an allosterically regulated enzyme of the Vmax type. It is a tetramer of identical subunits and each subunit is made up of three identifiable domains, the cofactor binding domain, the substrate binding domain, and the regulatory domain. Each subunit contacts two other subunits through adjacent cofactor binding domains and through adjacent regulatory domains. L-Serine, the physiological effector, inhibits catalytic activity by apparently tethering regulatory domains from adjacent subunits together through the formation of hydrogen bonds to each subunit. This investigation demonstrates that cross-linking adjacent regulatory domains with engineered disulfides produces catalytic inhibition in the absence of inhibitor in a manner similar to that produced by the inhibitor. The inhibition due to cross-linking can be completely reversed in a concentration dependent manner by dithiothreitol. The active mutant enzyme, containing the engineered cysteines in the reduced state, retains its ability to be inhibited by L-serine, although at a 100-fold higher concentration. Hill plots of the serine inhibition of mutant and native enzyme indicate that the number of interacting sites remains at 2 in the mutant enzyme. The reversible inhibition of enzyme activity that results from tethering adjacent regulatory domains with engineered disulfides suggests that these domains move in some manner relative to one another during the active to inhibited state transition. These observations support the model which predicts that catalytic activity is regulated by the movement of rigid domains about flexible hinges and that effector binding prevents this by locking the regulatory domains in a state that produces an open active site cleft.