The mechanism of velocity modulated allosteric regulation in D-3-phosphoglycerate dehydrogenase. Site-directed mutagenesis of effector binding site residues.

D-3-Phosphoglycerate dehydrogenase (EC 1.1.1.95) from Escherichia coli catalyzes the first committed step in serine biosynthesis and is allosterically regulated by L-serine, the end product of the pathway. Each subunit of the homotetramer is made up of three distinct domains with one of the intersubunit contacts being between adjacent regulatory domains. Each regulatory domain interface contains two symmetrical serine binding sites such that serine forms hydrogen bonds to both domains across the interface. Previous work (Al-Rabiee, R., Lee, E. J., and Grant, G. A. (1996) J. Biol. Chem. 271, 13013-13017) demonstrated that when adjacent regulatory domains are covalently linked to one another by engineered disulfide bonds, the enzyme was inactivated. Breaking the disulfide bonds by reduction restored enzymatic activity. This study demonstrates that the complementary situation is also true. Site-directed mutagenesis of three residues at the effector binding site, His344, Asn346, and Asn364', render the enzyme increasingly less susceptible to inhibition by the effector. When mutations result in a situation where it is no longer possible to establish a stable hydrogen bonding network across the regulatory domain interface, the inhibitory capacity of the effector is lost. Furthermore, mutations that produce as much as 5 orders of magnitude decrease in the ability of L-serine to inhibit the enzyme have no appreciable effect on the Km or kcat of the enzyme. These observations support the model that predicts that catalytic activity in D-3-phosphoglycerate dehydrogenase is regulated by the movement of adjacent regulatory domains about a flexible hinge and that effector binding tethers the regulatory domains together producing a state that results in a stable, open active site cleft that is no longer able to promote catalysis.