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

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-ofthe-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.D-3-Phosphoglycerate dehydrogenase (PGDH) 1 (EC 1.1.1.95) catalyzes the first committed step in the biosynthesis of Lserine (1-2). The enzyme displays normal Michaelis-Menten kinetics, but is inhibited in a cooperative allosteric manner by L-serine with a Hill coefficient of ϳ2 (1-5).PGDH is a tetramer composed of identical subunits that contain three distinct structural domains, the substrate binding domain, the nucleotide binding domain, and the regulatory or effector binding domain (6). The arrangement of subunits reveals that the enzyme can be viewed as a dimer of dimers. Contact between the nucleotide binding domains of 2 subunits form one dimer and contact between the regulatory domains of two of these dimers complete the tetramer contacts (see Fig. 1). L-Serine binds at the interface between adjacent regulatory domains as illustrated in Fig. 2. 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...

Identification of Amino Acid Residues Contributing to the Mechanism of Cooperativity in Escherichia coli d-3-Phosphoglycerate Dehydrogenase

“…Protein purity was assessed by SDS PAGE gels and all mutants were present as predominate single bands of the appropriate size. All mutants described in this study are based on PGDH 4CA (11) and were verified by automated sequencing on an Applied Biosystems 3730 DNA sequencer. PGDH activity was measured by following the decrease in absorbance at 340 nm in the presence of NADH and α-ketoglutarate.…”

“…Data were fitted to the Michaelis-Menton equation for the determination of K m and k cat and to the Hill equation for the determination of the serine concentration at 50% inhibition (I 0.5 ) and the Hill coefficient (11). Curve fitting was performed with Kaleidograph from Synergy Software.…”

“…Site-directed mutagenesis was performed and mutated proteins were expressed and isolated as previously described (11,13). Protein purity was assessed by SDS PAGE gels and all mutants were present as predominate single bands of the appropriate size.…”

“…The mechanism of allosteric regulation of E. coli PGDH by means of effector binding to its regulatory domain has been the subject of extensive investigation (2,3,10,11). However, it has not been until the recent elucidation of the crystal structure of native E. coli PGDH in the absence of L-serine (12) (PDB code: 1YBA) that direct comparison between the inhibited and active structures of this enzyme could be made.…”

D-3-Phosphoglycerate Dehydrogenase from Mycobacterium tuberculosis Is a Link between the Escherichia coli and Mammalian Enzymes

“…Third bar, some organisms also contain a PGDH that is devoid of a regulatory domain segment. They exist as dimers and may or may not retain Trp the Hill equation as previously described (17). Binding of L-serine to M. tuberculosis PGDH was performed as described previously (18) using tritiated L-serine.…”