Hybrid Tetramers Reveal Elements of Cooperativity in Escherichia colid-3-Phosphoglycerate Dehydrogenase

Abstract

D-3-Phosphoglycerate dehydrogenase from Escherichia coli is a tetramer of identical subunits that is inhibited when L-serine binds at allosteric sites between subunits. Co-expression of two genes, the native gene containing a charge difference mutation and a gene containing a mutation that eliminates serine binding, produces hybrid tetramers that can be separated by ion exchange chromatography. Activity in the hybrid tetramer with only a single intact serine binding site is inhibited by ϳ58% with a Hill coefficient of 1. Thus, interaction at a single regulatory domain interface does not, in itself, lead to the positive cooperativity of inhibition manifest in the native enzyme. Tetramers with only two intact serine binding sites purify as a mixture that displays a maximum inhibition level that is less than that of native enzyme, suggesting the presence of a population of tetramers that are unable to be fully inhibited. Differential analysis of this mixture supports the conclusion that it contains two forms of the tetramer. One form contains two intact serine binding sites at the same interface and is not fully inhibitable. The second form is a fully inhibitable population that has one serine binding site at each interface. Overall, the hybrid tetramers show that the positive cooperativity observed for serine binding is mediated across the nucleotide binding domain interface, and the negative cooperativity is mediated across the regulatory domain interface. That is, they reveal a pattern in which the binding of serine at one interface leads to negative cooperativity of binding of a subsequent serine at the same interface and positive cooperativity of binding of a subsequent serine to the opposite interface. This trend is propagated to subsequent binding sites in the tetramer such that the negative cooperativity that is originally manifest at one interface is decreased by subsequent binding of ligand at the opposite interface.

Quantitative Relationships of Site to Site Interaction in Escherichia coli d-3-Phosphoglycerate Dehydrogenase Revealed by Asymmetric Hybrid Tetramers

“…Whereas some insight into the interrelationship of the serine binding sites to each other has been gained from previous studies (5,7,18), much less has been discovered about the functional relationship of the active sites to the serine binding sites. The hybrid tetramers described here provide, for the first time, directly interpretable results that elucidate this relationship.…”

“…Production of Hybrid Tetramers by Co-expression-Hybrid tetramers were produced by the co-expression of two genes for PGDH within the same plasmid as described previously (7). In this study, one of the PGDH genes (designated Gene 1 in native amino-terminal sequence with a selected functional mutation.…”

“…The recent production of hybrid tetramers of PGDH by coexpression of two different PGDH genes has made it possible to start investigating the site to site interaction in these cooperative regulatory processes using asymmetric tetramers (7). This method previously demonstrated that the negative cooperativity of serine binding involved sites at the same regulatory domain interface, whereas the positive cooperativity of serine binding involved sites at opposite regulatory domain interfaces.…”

“…This amelioration of cooperativity is not seen with NAD ϩ . It has been proposed that the negative cooperativity of serine binding could result if a single effector molecule was capable of stabilizing the association of the two domains to the extent that it could exclude, or partially exclude, the binding of the second ligand at that interface (7).…”

“…There are two such interfaces at opposite ends of the tetramer and 2 serine molecules bind in each interface forming hydrogen-bonding contacts across the interface. It has been proposed that the binding of L-serine stabilizes the regulatory domain interface contacts and inhibits the active site by imparting rigidity to the molecule (7).…”