D-3-Phosphoglycerate dehydrogenase (PGDH) from Mycobacterium tuberculosis has been isolated to homogeneity and displays an unusual relationship to the Escherichia coli and mammalian enzymes. In almost all aspects investigated, the M. tuberculosis enzyme shares the characteristics of the mammalian PGDHs. These include an extended C-terminal motif, substrate inhibition kinetics, dependence of activity levels and stability on ionic strength, and the inability to utilize ␣-ketoglutarate as a substrate. The unique property that the M. tuberculosis enzyme shares with E. coli PGDH that it is very sensitive to inhibition by L-serine, with an I 0.5 ‫؍‬ 30 M. The mammalian enzymes are not inhibited by L-serine. In addition, the cooperativity of serine inhibition appears to be modulated by chloride ion, becoming positively cooperative in its presence. This is modulated by the gain of cooperativity in serine binding for the first two effector sites. The basis for the chloride modulation of cooperativity is not known, but the sensitivity to serine inhibition can be explained in terms of certain amino acid residues in critical areas of the structures. The differential sensitivity to serine inhibition by M. tuberculosis and human PGDH may open up interesting possibilities in the treatment of multidrug-resistant tuberculosis.D-3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) 1 has been investigated in a variety of organisms (1-4) and is a member of a family of proteins classified as 2-hydroxyacid dehydrogenases that are generally specific for substrates with a D-configuration (5). PGDH catalyzes the first committed step in the phosphorylated pathway of L-serine biosynthesis by converting D-3-phosphoglycerate to hydroxypyruvic acid phosphate utilizing NAD ϩ as a cofactor (1, 6). Subsequently, hydroxypyruvic acid phosphate is converted to phosphoserine by phosphoserine transaminase and then to L-serine by phosphoserine phosphatase (6). The reaction catalyzed by PGDH occurs spontaneously in vitro in the direction of conversion of hydroxypyruvic acid phosphate to phosphoglycerate.In some organisms, such as Escherichia coli (7), PGDH is strongly inhibited by L-serine (I 0.5 ϭ ϳ2-4 M), the end product of the pathway. E. coli PGDH is the most studied and is classified as a V-type enzyme (8) because L-serine regulates catalysis by altering the velocity of the reaction rather than the affinity of the substrate. In Bacillus subtilis, inhibition of PGDH by L-serine is less sensitive (I 0.5 ϳ 0.6 mM) but appears to lose its sensitivity to L-serine under oxidizing conditions (9). PGDH from Corynebacterium glutamicum has also been reported to be inhibited by L-serine only at very high concentrations (I 0.5 ϳ 10 mM) but has not been studied in homogeneous form (10). In addition, L-serine inhibition of both the B. subtilis and C. glutamicum PGDH require extensive preincubation of the enzyme with the inhibitor before appreciable inhibition can be measured. In the pea (Pisum sativum), the sensitivity to L-serine has been reported to be cold ...

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...

D-3-Phosphoglycerate dehydrogenase (PGDH) fromEscherichia coli is allosterically inhibited by L-serine, the end product of its metabolic pathway. Previous results have shown that inhibition by serine has a large effect on V max and only a small or negligible effect on K m . PGDH is thus classified as a V-type allosteric enzyme. In this study, the active site of PGDH has been studied by site-directed mutagenesis to assess the role of certain residues in substrate binding and catalysis. These consist of a group of cationic residues (Arg-240, Arg-60, Arg-62, Lys-39, and Lys-141) that potentially form an electrostatic environment for the binding of the negatively charged substrate, as well as the only tryptophan residue found in PGDH and which fits into a hydrophobic pocket immediately adjacent to the active site histidine residue. Interestingly, Trp-139 and Lys-141 are part of the polypeptide chain of the subunit that is adjacent to the active site. The results of mutating these residues show that Arg-240, Arg-60, Arg-62, and Lys-141 play distinct roles in the binding of the substrate to the active site. Mutants of Trp-139 show that this residue may play a role in stabilizing the catalytic center of the enzyme. Furthermore, these mutants appear to have a significant effect on the cooperativity of serine inhibition and suggest a possible role for Trp-139 in the cooperative interactions between subunits.

D-3-Phosphoglycerate dehydrogenase from Escherichia coli contains two Gly-Gly sequences that occur at junctions between domains. A previous study (Grant, G. A., Xu, X. L., and Hu, Z. (2000) Biochemistry 39, 7316 -7319) determined that the Gly-Gly sequence at the junction between the regulatory and substrate binding domain functions as a hinge between the domains. Mutations in this area significantly decrease the ability of serine to inhibit activity but have little effect on the K m and k cat . Conversely, the present study shows that mutations to the Gly-Gly sequence at the junction of the substrate and nucleotide binding domains, which form the active site cleft, have a significant effect on the k cat of the enzyme without substantially altering the enzyme's sensitivity to serine. In addition, mutation of Gly-294, but not Gly-295, has a profound effect on the cooperativity of serine inhibition. Interestingly, even though cooperativity of inhibition can be reduced significantly, there is little apparent effect on the cooperativity of serine binding itself. An additional mutant, G336V,G337V, also reduces the cooperativity of inhibition, but in this case serine binding also is reduced to the point at which it cannot be measured by equilibrium dialysis. The double mutant G294V,G336V demonstrates that strain imposed by mutation at one hinge can be relieved partially by mutation at the other hinge, demonstrating linkage between the two hinge regions. These data show that the two cooperative processes, serine binding and catalytic inhibition, can be uncoupled. Consideration of the allowable torsional angles for the side chains introduced by the mutations yields a range of values for these angles that the glycine residues likely occupy in the native enzyme. A comparison of these values with the torsional angles found for the inhibited enzyme from crystal coordinates provides potential beginning and ending orientations for the transition from active to inhibited enzyme, which will allow modeling of the dynamics of domain movement.

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.