THE MECHANISM OF VELOCITY MODULATED ALLOSTERIC REGULATION IN D-3-PHOSPHOGLYCERATE DEHYDROGENASE - CROSS-LINKING ADJACENT REGULATORY DOMAINSWITH ENGINEERED DISULFIDES MIMICS EFFECTOR-BINDING

D-3-Phosphoglycerate dehydrogenase (PGDH) (EC 1.1.1.95) from Escherich ia coli is an allosterically regulated enzyme of the V-max type, It is a tetramer of identical subunits and each subunit is made up of three identifiable domains, the cofactor binding domain, the substrate bind ing domain, and the regulatory domain, Each subunit contacts two other subunits through adjacent cofactor binding domains and through adjace nt regulatory domains, L-Serine, the physiological effector, inhibits catalytic activity by apparently tethering regulatory domains from adj acent subunits together through the formation of hydrogen bonds to eac h subunit, This investigation demonstrates that cross-linking adjacent regulatory domains with engineered disulfides produces catalytic inhi bition in the absence of inhibitor in a manner similar to that produce d by the inhibitor, The inhibition due to cross-linking can be complet ely reversed in a concentration dependent manner by dithiothreitol, Th e active mutant enzyme, containing the engineered cysteines in the red uced state, retains its ability to be inhibited by L-serine, although at a 100-fold higher concentration, Hill plots of the serine inhibitio n 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 domain s with engineered disulfides suggests that these domains move in some manner relative to one another during the active to inhibited state tr ansition, These observations support the model which predicts that cat alytic activity is regulated by the movement of rigid domains about fl exible hinges and that effector binding prevents this by locking the r egulatory domains in a state that produces an open active site cleft.