A MODEL FOR THE REGULATION OF D-3-PHOSPHOGLYCERATE DEHYDROGENASE, A V-MAX-TYPE ALLOSTERIC ENZYME

Escherichia coli D-3-phosphoglycerate dehydrogenase (ePGDH) is a tetra mer of identical subunits that is allosterically inhibited by L-serine , the end product of its metabolic pathway. Because serine binding aff ects the velocity of the reaction and not the binding of substrate or cofactor, the enzyme is classified as of the V-max type. Inhibition by a variety of amino acids and analogues of L-serine indicate that all three functional groups of serine are required for optimal interaction . Removing or altering any one functional group results in an increase in inhibitory concentration From micromolar to millimolar, and remova l or alteration of any two functional groups removes all inhibitory ab ility; Kinetic studies indicate at least two serine-binding sites, but the crystal structure solved in the presence of bound serine and dire ct serine-binding studies show that there are a total of four serine-b inding sites on the enzyme. However, approximately 85% inhibition is a ttained when only two sites are occupied. The three-dimensional struct ure of ePGDH shows that the serine-binding sites reside at the interfa ce between regulatory domains of adjacent subunits. Two serine molecul es bind at each of the two regulatory domain interfaces in the enzyme. When all four of the serines are bound, 100% inhibition of activity i s seen. However, because the domain contacts are symmetrical, the bind ing of only one serine at each interface is sufficient to produce appr oximately 85% inhibition. The tethering of the regulatory domains to e ach other through multiple hydrogen bonds from serine to each subunit apparently prevents the body of these domains from undergoing the reor ientation that must accompany a catalytic cycle. It is suggested that parr of the conformational change may involve a hinge formed in the vi cinity of the union of two antiparallel beta-sheets in the regulatory domains. The tethering function of serine, in turn, appears to prevent the substrate-binding domain from closing the cleft between it and th e nucleotide-binding domain, which may be necessary to form a producti ve hydrophobic environment for hydride transfer. Thus, the structure p rovides a plausible model that is consistent with the binding and inhi bition data and that suggests that catalysis and inhibition in this ra re V-max-type allosteric enzyme is based on the movement of rigid doma ins about flexible hinges.