Determinants of substrate specificity in D-3-phosphoglycerate dehydrogenase. Conversion of the M. tuberculosis enzyme from one that does not use α-ketoglutarate as a substrate to one that does.

d-3-Phosphoglycerate dehydrogenase (PGDH) converts d-3-phosphoglycerate (PGA) to phosphohydroxypyruvate (PHP) in the first step of l-serine biosynthesis. This reaction is reversible, and some PGDHs are capable of using α-ketoglutarate (αKG) instead of PHP in the reverse direction to produce α-hydroxyglutarate. The enzymes so far shown to have this ability are Type II PGDHs, suggesting that this may be a common feature of the Type II enzymes. Type I PGDHs examined so far do not share this feature. Inspection of PGDH sequences shows that a GCFCI … WXKX motif is commonly found in Type II PGDHs while a GRAGT … WXRX motif is commonly associated with Type I PGDHs. The removal of the cationic side chain at the first position shown above in the Type I PGDH from Mycobacterium tuberculosis converts it to an enzyme capable of using αKG where the native enzyme is not. It also produces an enzyme that regenerates NAD+ in the forward reaction when coupled to phosphoserine aminotransferase, as was previously shown for E. coli PGDH. Substitution of an arginyl residue for a lysyl residue at the second position of ecPGDH, decreases the kcat/Km of the enzyme by approximately 50-fold when using αKG, but only approximately 3-fold when using PHP. This suggests that a PGDH dependent cycle that conserves NAD+ in E. coli may be operative in many other organisms expressing the GCFCI … WXKX motif.

The many faces of partial inhibition: Revealing imposters with graphical analysis.

Partial inhibition occurs when an enzyme-inhibitor complex is capable of producing a product, as opposed to complete inhibition where the enzyme-inhibitor complex is completely inactive and cannot produce a product. While not frequently encountered, partial inhibition is also not a rare phenomenon and can potentially have significant repercussions later on in a research project. It is therefore important to recognize partial inhibition when it occurs. However, it cannot be recognized from the standard velocity versus substrate concentration plots or even from other primary plots such as Lineweaver-Burk plots. Fortunately, partial inhibition is easily identified by using replots which can be generated from the data already gathered to produce the primary plots. Partial inhibition phenomena are also not usually addressed in introductory textbooks and are often given only brief mention in some articles or books dealing with enzyme kinetics. The same types of approaches can be used to study enzyme activation phenomena, which can be considered the flip-side of inhibition. This review presents a graphical approach to identifying partial inhibition in an attempt to provide a comprehensive treatment for those wishing for or needing additional information in carrying out steady-state kinetic inhibition analyses.

Elucidation of a Self-Sustaining Cycle in Escherichia coli l-Serine Biosynthesis That Results in the Conservation of the Coenzyme, NAD.

The equilibrium of the reaction catalyzed by d-3-phosphoglycerate dehydrogenase (PGDH), the first enzyme in the l-serine biosynthetic pathway, is far in the direction away from serine synthesis. As such, the enzyme is usually assayed in this direction. To easily assay it in the direction of l-serine synthesis, it can be coupled to the next enzyme in the pathway, phosphoserine aminotransferase (PSAT), with the activity monitored by the conversion of NAD+ to NADH by PGDH. However, when PGDHs from several different species were coupled to PSAT, it was found that one of them, ecPGDH, conserves the coenzyme in the production of l-serine by utilizing an intrinsic cycle of NAD+/NADH interconversion coupled with the conversion of α-ketoglutarate (αKG) to α-hydroxyglutarate. Furthermore, the cycle can be maintained by production of αKG by the second enzyme in the pathway, PSAT, and does not require any additional enzymes. This is not the case for PGDH from another bacterial source, Mycobacterium tuberculosis, and a mammalian source, human liver, where net consumption of NAD+ occurs. Both NAD+ and NADH appear to remain tightly bound to ecPGDH during the cycle, effectively removing a requirement for the presence of an exogenous coenzyme pool to maintain the pathway and significantly reducing the energy requirement needed to maintain this major metabolic pathway.

D-3-Phosphoglycerate Dehydrogenase.

l-Serine is the immediate precursor of d-serine, a major agonist of the N-methyl-d-aspartate (NMDA) receptor. l-Serine is a pivotal amino acid since it serves as a precursor to a large number of essential metabolites besides d-serine. In all non-photosynthetic organisms, including mammals, a major source of l-serine is the phosphorylated pathway of l-serine biosynthesis. The pathway consists of three enzymes, d-3-phosphoglycerate dehydrogenase (PGDH), phosphoserine amino transferase (PSAT), and l-phosphoserine phosphatase (PSP). PGDH catalyzes the first step in the pathway by converting d-3-phosphoglycerate (PGA), an intermediate in glycolysis, to phosphohydroxypyruvate (PHP) concomitant with the reduction of NAD+. In some, but not all organisms, the catalytic activity of PGDH can be regulated by feedback inhibition by l-serine. Three types of PGDH can be distinguished based on their domain structure. Type III PGDHs contain only a nucleotide binding and substrate binding domain. Type II PGDHs contain an additional regulatory domain (ACT domain), and Type I PGDHs contain a fourth domain, termed the ASB domain. There is no consistent pattern of domain content that correlates with organism type, and even when additional domains are present, they are not always functional. PGDH deficiency results in metabolic defects of the nervous system whose systems range from microcephaly at birth, seizures, and psychomotor retardation. Although deficiency of any of the pathway enzymes have similar outcomes, PGDH deficiency is predominant. Dietary or intravenous supplementation with l-serine is effective in controlling seizures but has little effect on psychomotor development. An increase in PGDH levels, due to overexpression, is also associated with a wide array of cancers. In culture, PGDH is required for tumor cell proliferation, but extracellular l-serine is not able to support cell proliferation. This has led to the hypothesis that the pathway is performing some function related to tumor growth other than supplying l-serine. The most well-studied PGDHs are bacterial, primarily from Escherichia coli and Mycobacterium tuberculosis, perhaps because they have been of most interest mechanistically. However, the relatively recent association of PGDH with neuronal defects and human cancers has provoked renewed interest in human PGDH.

Regulatory Mechanism of Mycobacterium tuberculosis Phosphoserine Phosphatase SerB2.

Almost all organisms contain the same biosynthetic pathway for the synthesis of l-serine from the glycolytic intermediate, d-3-phosphoglycerate. However, regulation of this pathway varies from organism to organism. Many organisms control the activity of the first enzyme in the pathway, d-3-phosphoglycerate dehydrogenase (PGDH), by feedback inhibition through the interaction of l-serine with the ACT domains within the enzyme. The last enzyme in the pathway, phosphoserine phosphatase (PSP), has also been reported to be inhibited by l-serine. The high degree of sequence homology between Mycobacterium tuberculosis PSP (mtPSP) and Mycobacterium avium PSP (maPSP), which has recently been shown to contain ACT domains, suggested that the mtPSP also contained ACT domains. This raised the question of whether the ACT domains in mtPSP played a functional role similar to that of the ACT domains in PGDH. This investigation reveals that l-serine allosterically inhibits mtPSP by a mechanism of partial competitive inhibition by binding to the ACT domains. Therefore, in mtPSP, l-serine is an allosteric feedback inhibitor that acts by decreasing the affinity of the substrate for the enzyme. mtPGDH is also feedback inhibited by l-serine, but only in the presence of millimolar concentrations of phosphate. Therefore, the inhibition of mtPSP by l-serine would act as a secondary control point for the regulation of the l-serine biosynthetic pathway under physiological conditions where the level of phosphate would be below that needed for l-serine feedback inhibition of mtPGDH.

Modification of Cysteine.

This unit describes a number of methods for modifying cysteine residues of proteins and peptides. A general procedure for alkylation of cysteine residues in a protein of known size and composition with haloacyl reagents or N-ethylmaleimide (NEM) is presented, and alternate protocols describe similar procedures for use when the size and composition are not known and when only very small amounts of protein are available. Alkylations that introduce amino groups using bromopropylamine and N-(iodoethyl)-trifluoroacetamide are also presented. Two procedures that are often used for subsequent sequence analysis of the protein, alkylation with 4-vinylpyridine and acrylamide, are described, and a specialized procedure for 4-vinylpyridine alkylation of protein that has been adsorbed onto a sequencing membrane is also presented. Reversible modification of cysteine residues by way of sulfitolysis is described, and a protocol for oxidation with performic acid for amino acid compositional analysis is also provided. Gentle oxidation of cysteine residues to disulfides by exposure to air is described. Support protocols are included for recrystallization of iodoacetic acid, colorimetric detection of free sulfhydryls, and desalting of modified samples. © 2017 by John Wiley & Sons, Inc.

Mutagenic and chemical analyses provide new insight into enzyme activation and mechanism of the type 2 iron-sulfur l-serine dehydratase from Legionella pneumophila.

The crystal structure of the Type 2 l-serine dehydratase from Legionella pneumophila (lpLSD), revealed a "tail-in-mouth" configuration where the C-terminal residue acts as an intrinsic competitive inhibitor. This pre-catalytic structure undergoes an activation step prior to catalytic turnover. Mutagenic analysis of residues at or near the active site cleft is consistent with stabilization of substrate binding by many of the same residues that interact with the C-terminal cysteine and highlight the critical role of certain tail residues in activity. pH-rate profiles show that a residue with pK of 5.9 must be deprotonated and a residue with a pK of 8.5 must be protonated for activity. This supports an earlier suggestion that His 61 is the likely catalytic base. An additional residue with a pK of 8.5-9 increases cooperativity when it is deprotonated. This investigation also demonstrates that the Fe-S dehydratases convert the enamine/imine intermediates of the catalytic reaction to products on the enzyme prior to release. This is in contrast to pyridoxyl 5' phosphate based dehydratases that release an enamine/imine intermediate into solution, which then hydrolyzes to produce the ketoamine product.

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Unique Conformational and Chemical Transformations Occurring upon [4Fe-4S] Cluster Binding in the Type 2 L-Serine Dehydratase from Legionella pneumophila.

The type 2 L-serine dehydratase from Legionella pneumophila (lpLSD) contains a [4Fe-4S](2+) cluster that acts as a Lewis acid to extract the hydroxyl group of L-serine during the dehydration reaction. Surprisingly, the crystal structure shows that all four of the iron atoms in the cluster are coordinated with protein cysteinyl residues and that the cluster is buried and not exposed to solvent. If the crystal structure of lpLSD accurately reflects the structure in solution, then substantial rearrangement at the active site is necessary for the substrate to enter. Furthermore, repair of the oxidized protein when the cluster has degraded would presumably entail exposure of the buried cysteine ligands. Thus, the conformation required for the substrate to enter may be similar to those required for a new cluster to enter the active site. To address this, hydrogen-deuterium exchange combined with mass spectrometry (HDX MS) was used to probe the conformational changes that occur upon oxidative degradation of the Fe-S cluster. The regions that show the most significant differential HDX are adjacent to the cluster location in the holoenzyme or connect regions that are adjacent to the cluster. The observed decrease in flexibility upon cluster binding provides direct evidence that the "tail-in-mouth" conformation observed in the crystal structure also occurs in solution and that the C-terminal peptide is coordinated to the [4Fe-4S] cluster in a precatalytic conformation. This observation is consistent with the requirement of an activation step prior to catalysis and the unusually high level of resistance to oxygen-induced cluster degradation. Furthermore, peptide mapping of the apo form under nonreducing conditions revealed the formation of disulfide bonds between C396 and C485 and possibly between C343 and C385. These observations provide a picture of how the cluster loci are stabilized and poised to receive the cluster in the apo form and the requirement for a reduction step during cluster formation.

Comparison of Type 1 D-3-phosphoglycerate dehydrogenases reveals unique regulation in pathogenic Mycobacteria.

D-3-phosphoglycerate dehydrogenases (PGDH) from all organisms catalyze the conversion of D-3-phosphoglycerate to phosphohydroxypyruvate as the first step in the biosynthesis of l-serine. This investigation compares the properties of Type 1 PGDHs from seven different species and demonstrates that conserved residues in the ACT and ASB domains of some allow l-serine to act as a feedback inhibitor at low micromolar concentrations. In addition, the serine sensitivity is dependent on the presence of phosphate ions. These residues are most highly conserved among PGDHs from the actinomycetales family, but only certain pathogenic mycobacteria appear to have the full complement of residues required for high sensitivity to serine. These basic residues are also responsible for the presence of dual pH optima in the acidic region that is also phosphate dependent. Analytical ultracentrifugation analysis demonstrates that the dual pH optima do not require changes in oligomeric state. This study also demonstrates that substrate inhibition is a common feature of Type 1 PGDHs and that it is suppressed by phosphate, indicating that phosphate likely interacts at both the catalytic and regulatory sites. The unique features resulting from the complement of basic residues conserved in pathogenic mycobacteria may impart important metabolic advantages to these organisms.

Structure of L-serine dehydratase from Legionella pneumophila: novel use of the C-terminal cysteine as an intrinsic competitive inhibitor.

Here we report the first complete structure of a bacterial Fe-S l-serine dehydratase determined to 2.25 Å resolution. The structure is of the type 2 l-serine dehydratase from Legionella pneumophila that consists of a single polypeptide chain containing a catalytic α domain and a ß domain that is structurally homologous to the "allosteric substrate binding" or ASB domain of d-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis. The enzyme exists as a dimer of identical subunits, with each subunit exhibiting a bilobal architecture. The [4Fe-4S](2+) cluster is bound by residues from the C-terminal α domain and is situated between this domain and the N-terminal ß domain. Remarkably, the model reveals that the C-terminal cysteine residue (Cys 458), which is conserved among the type 2 l-serine dehydratases, functions as a fourth ligand to the iron-sulfur cluster producing a "tail in mouth" configuration. The interaction of the sulfhydryl group of Cys 458 with the fourth iron of the cluster appears to mimic the position that the substrate would adopt prior to catalysis. A number of highly conserved or invariant residues found in the ß domain are clustered around the iron-sulfur center. Ser 16, Ser 17, Ser 18, and Thr 290 form hydrogen bonds with the carboxylate group of Cys 458 and the carbonyl oxygen of Glu 457, whereas His 19 and His 61 are poised to potentially act as the catalytic base required for proton extraction. Mutation of His 61 produces an inactive enzyme, whereas the H19A protein variant retains substantial activity, suggesting that His 61 serves as the catalytic base. His 124 and Asn 126, found in an HXN sequence, point toward the Fe-S cluster. Mutational studies are consistent with these residues either binding a serine molecule that serves as an activator or functioning as a potential trap for Cys 458 as it moves out of the active site prior to catalysis.

Regulation of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase by phosphate-modulated quaternary structure dynamics and a potential role for polyphosphate in enzyme regulation.

D-3-phosphoglycerate dehydrogenase (PGDH) catalyzes the first reaction in the "phosphorylated" pathway of l-serine biosynthesis. In Mycobacterium tuberculosis, it is a type 1 enzyme (mtPGDH) in that it contains both an ACT domain and an ASB domain in addition to a catalytic domain. The published crystal structures (Protein Data Bank entries 1YGY and 3DC2) show a tartrate molecule interacting with cationic residues at the ASB-ACT domain interfaces and a serine molecule bound at the ACT domain interface. These sites have previously been shown to be involved in the mechanism of serine and substrate inhibition of catalytic activity. This investigation has revealed a mechanism of allosteric quaternary structure dynamics in mtPGDH that is modulated by physiologically relevant molecules, phosphate and polyphosphate. In the absence of phosphate and polyphosphate, the enzyme exists in equilibrium between an inactive dimer and an active tetramer that is insensitive to inhibition of catalytic activity by L-serine. Phosphate induces a conversion to an active tetramer and octamer that are sensitive to inhibition of catalytic activity by L-serine. Small polyphosphates (pyrophosphate and triphosphate) induce a conversion to an active dimer that is insensitive to L-serine inhibition. The difference in the tendency of each respective dimer to form a tetramer as well as slightly altered elution positions on size exclusion chromatography indicates that there is likely a conformational difference between the serine sensitive and insensitive states. This appears to constitute a unique mechanism in type 1 PGDHs that may be unique in pathogenic Mycobacterium species and may provide the organisms with a unique metabolic advantage.

Identification and characterization of two new types of bacterial L-serine dehydratases and assessment of the function of the ACT domain.

Two new types of bacterial Fe-S L-serine dehydratases have been identified. These join two previously recognized enzyme types, for a total of four, that are distinguished on the basis of domain arrangement and amino acid sequence. A Type 3 enzyme from Amphibacillus xylanus (axLSD) and a Type 4 enzyme from Heliscomenobacter hydrossis (hhLSD) were cloned, expressed, purified, and characterized. Like the Type 1 enzyme from Bacillus subtilis (bsLSD), axLSD required a monovalent cation, preferably potassium, for activity. However, the hhLSD was without activity even after reconstitution of the iron-sulfur center by a process that successfully restored activity to oxygen-inactivated axLSD. This and other characteristics suggest that this Type 4 protein may be a pseudoenzyme. The oxygen sensitivity of axLSD was greater than other L-serine dehydratases so far studied and suggested that there may be significant conformational differences among the four types resulting in widely different solvent accessibility of the Fe-S clusters in these enzymes. The role of the ACT domain in these enzymes was explored by deleting it from bsLSD. Although there was an effect on the kinetic parameters, this domain was not responsible for the cation requirement nor did its removal have a significant effect on oxygen sensitivity.

Kinetic evidence of a noncatalytic substrate binding site that regulates activity in Legionella pneumophila L-serine dehydratase.

The L-serine dehydratase from Legionella pneumophila (lpLSD) has recently been shown to contain a domain (ß domain) that has a high degree of structural homology with the ASB domain of d-3-phosphoglycerate dehydrogenase (PGDH) from Mycobacterium tuberculosis. Furthermore, this domain has been shown by sequence homology to be present in all bacterial L-serine dehydratases that utilize an Fe-S catalytic center. In PGDH, L-serine binds to the ACT domain to inhibit catalytic activity. However, substrate must be bound to the ASB domain for serine to exert its effect. As such, the ASB domain acts as a codomain for the action of L-serine. Pre-steady-state kinetic analysis of L-serine binding to lpLSD demonstrates that L-serine binds to a second noncatalytic site and produces a conformational change in the enzyme. The rate of this conformational change is too slow for its participation in the catalytic cycle but rather occurs prior to catalysis to produce an activated form of the enzyme. That the conformational change must occur prior to catalysis is shown by a lag in the production of product that exhibits essentially the same rate constant as the conformational change. The second, noncatalytic site for L-serine is likely to be the ASB domain (ß domain) of lpLSD that functions in a manner similar to that in PGDH. A mechanism whose overall effect is to keep L-serine levels from accumulating to high levels while not completely depleting the L-serine pool in the bacterial cell is proposed.

Allosteric activation and contrasting properties of L-serine dehydratase types 1 and 2.

Bacterial L-serine dehydratases differ from mammalian L- and D-serine dehydratases and bacterial D-serine dehydratases by the presence of an iron-sulfur center rather than a pyridoxyl phosphate prosthetic group. They exist in two forms, types 1 and 2, distinguished by their sequence and oligomeric configuration. Both types contain an ASB domain, and the type 1 enzymes also contain an ACT domain in a tandem arrangement with the ASB domain like that in type 1 D-3-phosphoglycerate dehydrogenases (PGDHs). This investigation reveals striking kinetic differences between L-serine dehydratases from Bacillus subtilis (bsLSD, type 1) and Legionella pneumophila (lpLSD, type 2). lpLSD is activated by monovalent cations and inhibited by monovalent anions. bsLSD is strongly activated by cations, particularly potassium, and shows a mixed response to anions. Flouride is a competitive inhibitor for lpLSD but an apparent activator for bsLSD at low concentrations and an inhibitor at high concentrations. The reaction products, pyruvate and ammonia, also act as activators but to different extents for each type. Pyruvate activation is competitive with L-serine, but activation of the enzyme is not compatible with it simply competing for binding at the active site and suggests the presence of a second, allosteric site. Because activation can be eliminated by higher levels of L-serine, it may be that this second site is actually a second serine binding site. This is consistent with type 1 PGDH in which the ASB domain functions as a second site for substrate binding and activation.

Contrasting catalytic and allosteric mechanisms for phosphoglycerate dehydrogenases.

D-3-Phosphoglycerate dehydrogenases (PGDH) exist with at least three different structural motifs and the enzymes from different species display distinctly different mechanisms. In many species, particularly bacteria, the catalytic activity is regulated allosterically through binding of l-serine to a distinct structural domain, termed the ACT domain. Some species, such as Mycobacterium tuberculosis, contain an additional domain, called the "allosteric substrate binding" or ASB domain, that functions as a co-domain in the regulation of catalytic activity. That is, both substrate and effector function synergistically in the regulation of activity to give the enzyme some interesting properties that may have physiological relevance for the persistent state of tuberculosis. Both enzymes function through a V-type regulatory mechanism and, in the Escherichia coli enzyme, it has been demonstrated that this results from a dead-end complex that decreases the concentration of active species rather than a decrease in the velocity of the active species. This review compares and contrasts what we know about these enzymes and provides additional insight into their mechanism of allosteric regulation.

Kinetic, mutagenic, and structural homology analysis of L-serine dehydratase from Legionella pneumophila.

A structural database search has revealed that the same fold found in the allosteric substrate binding (ASB) domain of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase (PGDH) is found in l-serine dehydratase from Legionella pneumophila. The M. tuberculosis PGDH ASB domain functions in the control of catalytic activity. Bacterial l-serine dehydratases are 4Fe-4S proteins that convert l-serine to pyruvate and ammonia. Sequence homology reveals two types depending on whether their α and ß domains are on the same (Type 2) or separate (Type 1) polypeptides. The α domains contain the catalytic iron-sulfur center while the ß domains do not yet have a described function, but the structural homology with PGDH suggests a regulatory role. Type 1 ß domains also contain additional sequence homologous to PGDH ACT domains. A continuous assay for l-serine dehydratase is used to demonstrate homotropic cooperativity, a broad pH range, and essential irreversibility. Product inhibition analysis reveals a Uni-Bi ordered mechanism with ammonia dissociating before pyruvate. l-Threonine is a poor substrate and l-cysteine and d-serine are competitive inhibitors with K(i) values that differ by almost 10-fold from those reported for Escherichia colil-serine dehydratase. Mutagenesis identifies the three cysteine residues at the active site that anchor the iron-sulfur complex.

Transient kinetic analysis of L-serine interaction with Escherichia coli D-3-phosphoglycerate dehydrogenase containing amino acid mutations in the hinge regions.

In Escherichia colid-3-phosphoglycerate dehydrogenase, the amino acid sequences G294-G295 and G336-G337 are found between structural domains and appear to function as hinge regions. Mutagenesis studies of these sequences showed that bulky side chains had significant effects on the kinetic properties of the enzyme. Placement of a tryptophanyl residue near the serine binding site (W139F/E360W) allows serine binding to be monitored by fluorescence quenching analysis. Pre-steady-state analysis has demonstrated that serine binds to two forms of the free enzyme, E and E*. Conversion of Gly-336 to valine has its main effect on the Kd of serine binding to one form of the free enzyme (E) while maintaining the cooperativity of binding observed in the native enzyme. Conversion of Gly-294 to valine eliminates a rate limiting conformational change that follows serine binding to E. The conformational change between the two forms of free enzyme is maintained, but the Hill coefficient for cooperativity is significantly lowered. The data indicate that the cooperative transmission induced by serine binding is transmitted through the Gly294-Gly295 hinge region to the opposite serine binding interface and that this is most likely propagated by way of the substrate binding domain-regulatory domain interface. In the G294 mutant enzyme, both serine bound species, E·Ser and E*·Ser, are present in significant amounts indicating that cooperativity of serine binding does not result from the binding to two different forms. The data also suggest that the E* form may be inactive even when serine is not bound.

Transient kinetic analysis of the interaction of L-serine with Escherichia coli D-3-phosphoglycerate dehydrogenase reveals the mechanism of V-type regulation and the order of effector binding.

Pre-steady state stopped-flow analysis of Escherichia coli d-3-phosphoglycerate dehydrogenase (PGDH) reveals that the physiological inhibitor, l-serine, exerts its effect on at least two steps in the kinetic mechanism, but to very different degrees. First, there is a small but significant effect on the dissociation constant of NADH, the first substrate to bind in the ordered mechanism. The effect of serine is mainly on the binding off rate, increasing the K(d) to 5 and 23 muM from 0.6 and 9 muM, respectively, for the two sets of sites in the enzyme. A more profound effect is seen after the second substrate is added. Serine reduces the amplitude of the signal without a significant effect on the observed rate constants for binding. The serine concentration that reduces the amplitude by 50% is equal to the K(0.5) for serine inhibition. The data are consistent with the conclusion that serine binding eliminates a conformational change subsequent to substrate binding by formation of a dead-end quaternary complex consisting of enzyme, coenzyme, substrate, and effector. Thus, the mechanistic basis for V-type regulation in this enzyme is a reduction in the population of active species rather than a differential decrease in the velocity of active species. Pre-steady state analysis of binding of serine to a mutant PGDH (W139F/E360W) demonstrates that each serine binding interface produces an integrated fluorescent signal. The observed rate data are complex but conform to a model in which serine can bind to two forms of the enzyme with different affinities. The integrated signal from each interface allows the amplitude data to clearly define the order of binding to each site, and modeling the amplitude data with species distribution equations clearly demonstrates an alternate interface binding mechanism and the direction of binding cooperativity.

Role of the anion-binding site in catalysis and regulation of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase.

D-3-Phosphoglycerate dehydrogenase from Mycobacterium tuberculosis displays substantial substrate inhibition in the direction of NADH oxidation by its physiological substrate, hydroxypyruvic acid phosphate (HPAP). Previous investigations showed that plots of substrate concentration versus activity derived from steady state assays could be fit with the equation for complete uncompetitive inhibition and that the mechanism may be allosteric. This investigation uses a simulation of transient kinetic data to demonstrate that the mechanism is consistent with the interaction of substrate at a second site called the anion-binding site. While addition of substrate at the active site is ordered, with HPAP binding before NADH, NADH can compete with the substrate for binding to the allosteric site and thereby eliminate the substrate inhibition. Fluorescence resonance energy transfer analysis of mutants with specific tryptophan residues converted to phenylalanine residues demonstrates that the main interaction of NADH with the enzyme, in the absence of substrate, is at the allosteric anion-binding site. This is further confirmed by mutations of basic residues at the anion-binding site which also demonstrates that these residues are necessary for inhibition by l-serine when it binds to the regulatory domain. This may indicate that a ligand must be bound to the anion-binding site for l-serine inhibition, providing a potential mechanism for low levels of activity in the presence of high levels of inhibitor.