Abstract
A set of asymmetric hybrid tetramers of Escherichia coli d-3-phosphoglycerate dehydrogenase (PGDH) have been made by gene co-expression and KSCN-induced dimer exchange. These tetramers contain varied numbers of active sites and effector binding sites arranged in different orientations within the tetramer. They reveal that PGDH displays half-of-the-sites activity with respect to its active sites and that the two sites that are active at any particular time lie in subunits on either side of the nucleotide binding domain interface. Half-of-the-sites functionality is also observed for the effector even though all four sites eventually bind effector. That is, only two effector sites need to be occupied for maximum inhibition. Binding of the last two effector molecules does not contribute functionally to inhibition of activity. Furthermore, positive cooperativity of inhibition of activity by the effector is completely dependent on the positive cooperativity of binding of the effector. Binding of the first effector molecule produces a conformational change that essentially completely inhibits the active site within the subunit to which it binds and produces an approximate 33% inhibition of the active site in the subunit to which it is not bound. Binding of the second effector at the opposite regulatory domain interface completes the inhibition of activity. This simple relationship defines the positional and quantitative influence of effector ligand binding on activity and can be used to predict the maximum level of inhibition of individual hybrid tetramers. In addition, the site-specific quantitative relationship of effector binding to individual active sites can be used to model the inhibition profile with relatively good agreement. These simple rules for the site to site interaction in PGDH provide significant new insight into the mechanism of allostery of this enzyme.
Highlights
D-3-Phosphoglycerate dehydrogenase (PGDH)1 (EC 1.1.1.95) catalyzes the first committed step in the biosynthesis of Lserine [1,2]
Hybrid tetramers of phosphoglycerate dehydrogenase (PGDH) can be produced in homogeneous form using two complementary approaches
Co-expression of two individual genes of PGDH results in association of subunits in all possible combinations not all can be obtained in homogeneous form after ion-exchange chromatography
Summary
D-3-Phosphoglycerate dehydrogenase (PGDH) (EC 1.1.1.95) catalyzes the first committed step in the biosynthesis of Lserine [1,2]. 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. Several reports have appeared that demonstrate that certain tetrameric proteins possessing the dimer of dimers motif can be dissociated into relatively stable dimers with KSCN if one interface is more stable than the other (9 –10) Application of this method to PGDH resulted in the production of homogeneous hybrid tetramers resulting from dimer dissociation and recombination.
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