A Novel Mechanism for Substrate Inhibition in Mycobacterium tuberculosis d-3-Phosphoglycerate Dehydrogenase

Abstract

Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase undergoes significant inhibition of activity with increasing concentrations of its substrate, hydroxypyruvic acid phosphate. The enzyme also displays an unusual dual pH optimum. A significant decrease in the K i for substrate inhibition at pH values corresponding to the valley between these optima is responsible for this phenomena. The change in K i has an average pK of ϳ5.8 and involves two functional groups that are protonated and two functional groups that are unprotonated for optimal substrate inhibition to occur. Mutagenesis of positively charged amino acid residues at a putative anion binding site previously revealed by the x-ray structure, produces significant changes in the pH-dependent profile of substrate inhibition. Several single residue mutations eliminate the dual pH optima by reducing substrate inhibition between pH 5 and 7 and a triple mutation was identified that eliminates the substrate inhibition altogether. The mutagenesis data support the conclusion that the anion binding site represents a new allosteric site for the control of enzyme activity and functions in a novel mechanism for substrate inhibition.D-3-Phosphoglycerate dehydrogenase (PGDH) 2 (EC 1.1.1.95) from Mycobacterium tuberculosis has been shown to differ markedly in structure and activity (1, 2) from the well studied enzyme from Escherichia coli (3). Like the E. coli enzyme, M. tuberculosis PGDH is a homotetramer, but unlike the E. coli enzyme, whose subunits are composed of three distinct domains, each M. tuberculosis subunit is composed of four distinct domains. The two enzymes have in common a nucleotide binding domain, a substrate binding domain, and a regulatory domain that appear to function similarly in both. The regulatory domains of these enzymes are members of the ACT domain family of small molecule binding regulatory domains (4 -6) that bind L-serine, an inhibitor of enzymatic activity. The fourth domain in M. tuberculosis PGDH is called the "intervening domain" and is located sequentially between the substrate binding domain and the regulatory domain. The function of this fourth domain in the PGDH subunit is not known but it forms what appears to be a new ligand binding site. This site is a small, solvent accessible pocket surrounded by positively charged amino acid side chains. It was first recognized from the crystal structure where it was observed to bind L-tartrate, a component of the crystallization buffer. Because L-tartrate is found only in plants, and it was present in the buffer in high concentrations, it is unlikely that it is the physiological ligand for this site. Therefore, for simplicity and to avoid ambiguity, we will refer to this site as the "anion binding site" because it appears to be composed completely of cationic side chains.E. coli PGDH exhibits activity with both the physiological substrate, hydroxypyruvic acid phosphate (HPAP), as well as ␣-ketoglutarate, an analog of HPAP. The M. tuberculosis enzyme, on the other hand, displays acti...

Removal of Substrate Inhibition and Increase in Maximal Velocity in the Short Chain Dehydrogenase/Reductase Salutaridine Reductase Involved in Morphine Biosynthesis

“…However, the removal of (or a decrease in) substrate inhibition is generally accompanied by reduced catalytic activity in many enzymes including lactate dehydrogenase, 17␤-hydroxysteroid dehydrogenase, D-phosphoglycerate dehydrogenase, sinapyl alcohol dehydrogenase, and tetrahydroquinone dehalogenase (39,(42)(43)(44)(45). Compared with wild-type SalR, the independent F104A and I275A mutants displayed weak substrate inhibition accompanied by similar or even increased reaction velocities.…”

“…2A (39). For sulfotransferases, the crystal structure showed that the substrate adopts a different conformation and binds closer to the cofactor binding site after the cofactor has been desulfated (40,41).…”

Structure-Based Mutational Studies of Substrate Inhibition of Betaine Aldehyde Dehydrogenase BetB from Staphylococcus aureus

“…The latter mechanism can be associated with the dehydrogenase residues located both in the substrate and in cofactor binding sites. Recent biochemical studies have demonstrated that substrate inhibition of several dehydrogenases can be significantly reduced or eliminated by single mutations in the enzyme active site, which can be accompanied by a reduction or increase in the reaction rate and usually correlates with a reduction of substrate affinity (increasing K m ) (4,7,10,13). For example, the mutation S163L in human lactate dehydrogenase eliminates substrate inhibition with a minor effect on its turnover rate, whereas in Bacillus stearothermophilus lactate dehydrogenase the D38R mutation reduces substrate inhibition 3-fold (4,14).…”

“…For dehydrogenases, several molecular mechanisms of substrate inhibition have been proposed, including the formation of a covalent adduct between the oxidized forms of substrate and cofactor, allosteric inhibition (which occurs away from the active site, e.g., in D-3-phosphoglycerol dehydrogenase from Mycobacterium tuberculosis), and the formation of a nonproductive enzyme complex with cofactor and/or substrate (6)(7)(8)(9)(10)(11)(12)(13). The latter mechanism can be associated with the dehydrogenase residues located both in the substrate and in cofactor binding sites.…”

Highly efficient enzymatic synthesis of tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate with a mutant alcohol dehydrogenase of Lactobacillus kefir

“…The kinetic parameters (K m , k cat , K is , k cat(i) ) of these three ADHs toward CDOH are summarized in Table 2. In the case of partially uncompetitive inhibition, the reaction rate at a relative high substrate concentration can be simply reduced to a supposed value of V i (Burton et al 2007;LiCata and Allewell 1997). In the current study, the value of k cat(i) , which is the catalytic constant related to V i , was treated as an important parameter for comparing the catalytic efficiency with the existence of substrate inhibition.…”