Allosteric coupling in pyruvate dehydrogenase kinase 2

Mitochondrial pyruvate dehydrogenase kinase 2 (PDHK2) phosphorylates the pyruvate dehydrogenase multienzyme complex (PDC) and thereby controls the rate of oxidative decarboxylation of pyruvate. The activity of PDHK2 is regulated by a variety of metabolites such as pyruvate, NAD (+), NADH, CoA, and acetyl-CoA. The inhibitory effect of pyruvate occurs through the unique binding site, which is specific for pyruvate and its synthetic analogue dichloroacetate (DCA). The effects of NAD (+), NADH, CoA, and acetyl-CoA are mediated by the binding site that recognizes the inner lipoyl-bearing domain (L2) of the dihydrolipoyl transacetylase (E2). Both allosteric sites are separated from the active site of PDHK2 by more than 20 A. Here we show that mutations of three amino acid residues located in the vicinity of the active site of PDHK2 (R250, T302, and Y320) make the kinase resistant to the inhibitory effect of DCA, thereby uncoupling the active site from the allosteric site. In addition, we provide evidence that substitutions of R250 and T302 can partially or completely uncouple the L2-binding site. Based on the available structural data, R250, T302, and Y320 stabilize the "open" and "closed" conformations of the built-in lid that controls the access of a nucleotide into the nucleotide-binding cavity. This strongly suggests that the mobility of ATP lid is central to the allosteric regulation of PDHK2 activity serving as a conformational switch required for communication between the active site and allosteric sites in the kinase molecule.     

Probing the mechanism of inactivation of human pyruvate dehydrogenase by phosphorylation of three sites

Activity of the mammalian pyruvate dehydrogenase complex (PDC) is regulated by phosphorylation-dephosphorylation of three serine residues (designated site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) in the alpha subunit of the pyruvate dehydrogenase (E1) component. Substitutions of the phosphorylation sites were generated by site-directed mutagenesis. Glutamate (S1E) and aspartate (S1D) substitutions at site 1 resulted in the complete loss of PDC activity; however, these mutants were variably active in the decarboxylation and 2,6-dichlorophenolindophenol assays. S1Q had only 3% of wild-type PDC activity. The apparent K(m) values for pyruvate increased for the mutants of site 1 when determined in the 2,6-dichlorophenolindophenol assay. The substitutions at sites 2 and 3 caused only moderate reductions in activity in the three assays. S3E had a 27-fold increase in the apparent K(m) for thiamine pyrophosphate and 8-fold increase in the K(i) for pyrophosphate. Site 3 was almost completely protected from phosphorylation by thiamine pyrophosphate. The results show that the size rather than negative charge of the substituted amino acid residue affects the active site of E1 and that modification of each of the three serine residues affect the active site in a site-specific manner for its ability to bind the cofactor and substrates.     

Histidine mutagenesis of Arabidopsis thaliana pyruvate dehydrogenase kinase.

Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Analysis of the primary amino-acid sequences of PDK from various sources reveals that these enzymes include the five domains characteristic of prokaryotic two-component His-kinases, despite the fact that PDK exclusively phosphorylates Ser residues in the E1alpha subunit of the PDC. This seeming contradiction might be resolved if the PDK-catalyzed reaction employed a phospho-His intermediate. The results from pH-stability studies of autophosphorylated Arabidopsis thaliana PDK did not provide any support for a phospho-His intermediate. Furthermore, site-directed mutagenesis of the two most likely phosphotransfer His residues (H121 and H168) did not abolish either PDK autophosphorylation or the ability to transphosphorylate E1alpha. Thus, PDK is a unique type of protein kinase having a His-kinase-like sequence but Ser-kinase activity.     

Crystal structure of an asymmetric complex of pyruvate dehydrogenase kinase 3 with lipoyl domain 2 and its biological implications

A homodimer of pyruvate dehydrogenase kinase (PDHK) is an integral part of pyruvate dehydrogenase complex (PDC) to which it is anchored primarily through the inner lipoyl-bearing domains (L2) of transacetylase component. The catalytic cycle of PDHK and its translocation over the PDC surface is thought to be mediated by the "symmetric" and "asymmetric" modes, in which the PDHK dimer binds to two and one L2-domain(s), respectively. Whereas the structure of the symmetric PDHK/L2 complex was reported, the structural organization and functional role of the asymmetric complex remain obscure. Here, we report the crystal structure of the asymmetric PDHK3/L2 complex that reveals several functionally important features absent from the previous structures. First, the PDHK3 subunits have distinct conformations: one subunit exhibits "open" and the other "closed" configuration of the putative substrate-binding cleft. Second, access to the closed cleft is additionally restricted by local unwinding of the adjacent alpha-helix. Modeling indicates that the target peptide might gain access to the PDHK active center through the open but not through the closed cleft. Third, the ATP-binding loop in one PDHK3 subunit adopts an open conformation, implying that the nucleotide loading into the active site is mediated by the inactive "pre-insertion" binding mode. Altogether our data suggest that the asymmetric complex represents a physiological state in which binding of a single L2-domain activates one of the PDHK protomers while inactivating another. Thus, the L2-domains likely act not only as the structural anchors but also modulate the catalytic cycle of PDHK.     

Role of pyruvate dehydrogenase kinase isoenzyme 4 (PDHK4) in glucose homoeostasis during starvation

The PDC (pyruvate dehydrogenase complex) is strongly inhibited by phosphorylation during starvation to conserve substrates for gluconeogenesis. The role of PDHK4 (pyruvate dehydrogenase kinase isoenzyme 4) in regulation of PDC by this mechanism was investigated with PDHK4-/- mice (homozygous PDHK4 knockout mice). Starvation lowers blood glucose more in mice lacking PDHK4 than in wild-type mice. The activity state of PDC (percentage dephosphorylated and active) is greater in kidney, gastrocnemius muscle, diaphragm and heart but not in the liver of starved PDHK4-/- mice. Intermediates of the gluconeogenic pathway are lower in concentration in the liver of starved PDHK4-/- mice, consistent with a lower rate of gluconeogenesis due to a substrate supply limitation. The concentration of gluconeogenic substrates is lower in the blood of starved PDHK4-/- mice, consistent with reduced formation in peripheral tissues. Isolated diaphragms from starved PDHK4-/- mice accumulate less lactate and pyruvate because of a faster rate of pyruvate oxidation and a reduced rate of glycolysis. BCAAs (branched chain amino acids) are higher in the blood in starved PDHK4-/- mice, consistent with lower blood alanine levels and the importance of BCAAs as a source of amino groups for alanine formation. Non-esterified fatty acids are also elevated more in the blood of starved PDHK4-/- mice, consistent with lower rates of fatty acid oxidation due to increased rates of glucose and pyruvate oxidation due to greater PDC activity. Up-regulation of PDHK4 in tissues other than the liver is clearly important during starvation for regulation of PDC activity and glucose homoeostasis.     

Roles of three well-conserved arginine residues in mediating the catalytic activity of tobacco acetohydroxy acid synthase

Acetohydroxy acid synthase (AHAS, EC 2.2.1.6; also known as acetolactate synthase, ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in plants and microorganisms. AHAS is the target of several classes of herbicides. In the present study, the role of three well-conserved arginine residues (R141, R372, and R376) in tobacco AHAS was determined by site-directed mutagenesis. The mutated enzymes, referred to as R141A, R141F, and R376F, were inactive and unable to bind to the cofactor, FAD. The inactive mutants had the same secondary structure as that of the wild type. The mutants R141K, R372F, and R376K exhibited much lower specific activities than the wild type, and moderate resistance to herbicides such as Londax, Cadre, and/or TP. The mutation R141K showed a strong reduction in activation efficiency by ThDP, while the mutations R372K and R376K showed a strong reductions in activation efficiency by FAD in comparison to the wild type enzyme. Taking into account the data presented here and the homology model constructed previously [Le et al. (2004) Biochem. Biophys. Res. Commun. 317, 930-938], it is suggested that the three amino acid residues studied (R141, R372, and R376) are located essentially at the enzyme active site, and, furthermore, that residues R372 and R376 are possibly responsible for the binding of the enzyme to FAD.     

Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism

Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.     

Involvement of alpha-cysteine-62 and beta-tryptophan-135 in human pyruvate dehydrogenase catalysis.

Pyruvate dehydrogenase (E1), a heterotetramer (alpha(2)beta(2)), is the first catalytic component of the mammalian pyruvate dehydrogenase complex (PDC). To investigate the roles of cysteine-62 of E1alpha (alphaC62) and tryptophan-135 of E1beta (betaW135) (identified previously as active site residues using chemical modifications) in E1 catalysis, two recombinant human E1 mutants were generated using site-directed mutagenesis: alphaC62A and betaW135L. Compared to wild-type, k(cat) values for alphaC62A and betaW135L measured by PDC assay were markedly reduced to 7.2 and 11. 6%, respectively. Apparent K(m) values for thiamin pyrophosphate (TPP) were increased approximately sixfold for both mutants, resulting in catalytic efficiency for TPP of only 1-2% of the wild-type E1. K(m) values for pyruvate increased only moderately (twofold). The alphaC62A and betaW135L mutants were less thermostable than wild-type E1. The conformations of the mutant apo-E1s determined by spectral analysis were different from that of the wild-type apo-E1. CD spectral analysis indicated that TPP binding was affected for both the alphaC62A and betaW135L mutant E1s. The substrate analogs, fluoropyruvate and bromopyruvate, were shown to be active site-directed inhibitors of human E1; in the absence of TPP, bromopyruvate (but not fluoropyruvate) inhibited human E1 due to SH-group modification. Pyruvate induced inactivation of human E1 could be restored by thiol reagents. Cysteine-62 (and maybe another group) is proposed to be involved in E1 inhibition by the substrate and substrate analogs. Taken together these results indicate that alphaC62 and betaW135 facilitate coenzyme binding, and alphaC62 could be near the substrate-binding site.     

Regulatory roles of the N-terminal domain based on crystal structures of human pyruvate dehydrogenase kinase 2 containing physiological and synthetic ligands

Pyruvate dehydrogenase kinase (PDHK) regulates the activity of the pyruvate dehydrogenase multienzyme complex. PDHK inhibition provides a route for therapeutic intervention in diabetes and cardiovascular disorders. We report crystal structures of human PDHK isozyme 2 complexed with physiological and synthetic ligands. Several of the PDHK2 structures disclosed have C-terminal cross arms that span a large trough region between the N-terminal regulatory (R) domains of the PDHK2 dimers. The structures containing bound ATP and ADP demonstrate variation in the conformation of the active site lid, residues 316-321, which enclose the nucleotide beta and gamma phosphates at the active site in the C-terminal catalytic domain. We have identified three novel ligand binding sites located in the R domain of PDHK2. Dichloroacetate (DCA) binds at the pyruvate binding site in the center of the R domain, which together with ADP, induces significant changes at the active site. Nov3r and AZ12 inhibitors bind at the lipoamide binding site that is located at one end of the R domain. Pfz3 (an allosteric inhibitor) binds in an extended site at the other end of the R domain. We conclude that the N-terminal domain of PDHK has a key regulatory function and propose that the different inhibitor classes act by discrete mechanisms. The structures we describe provide insights that can be used for structure-based design of PDHK inhibitors.     

Mutagenesis of non-conserved active site residues improves the activity and narrows the specificity of human thymidine kinase 2

Human thymidine kinase 2 (TK2) is critical for the nucleotide salvage pathway and phosphorylation of nucleoside analog prodrugs in vivo; however, it remains poorly studied because of difficulties in expressing it heterologously. TK2 is strictly pyrimidine-specific, whereas its phylogenetic relative, the Drosophila melanogaster deoxyribonucleoside kinase (DmdNK), shows higher activity and broader specificity towards both pyrimidines and purines. These differences are counterintuitive, as only two of 29 active site residues differ in the two enzymes: F80 and M118 in DmdNK are L78 and L116 in TK2. In addition to reporting an optimized protocol for the expression and purification of TK2, we have used site-directed mutagenesis to introduce the DmdNK-like amino acids into TK2, and characterized the three resulting enzymes (L78F-TK2, L116M-TK2, and L78F/L116M-TK2). These mutations improve the K(M) for thymidine, increasing the catalytic activity of L78F/L116M-TK2 4.4-fold, yet leaving the activity for deoxycytidine or the purine nucleosides unchanged.     

Crystal structure of pyruvate dehydrogenase kinase 3 bound to lipoyl domain 2 of human pyruvate dehydrogenase complex

The human pyruvate dehydrogenase complex (PDC) is regulated by reversible phosphorylation by four isoforms of pyruvate dehydrogenase kinase (PDK). PDKs phosphorylate serine residues in the dehydrogenase (E1p) component of PDC, but their amino-acid sequences are unrelated to eukaryotic Ser/Thr/Tyr protein kinases. PDK3 binds to the inner lipoyl domains (L2) from the 60-meric transacetylase (E2p) core of PDC, with concomitant stimulated kinase activity. Here, we present crystal structures of the PDK3-L2 complex with and without bound ADP or ATP. These structures disclose that the C-terminal tail from one subunit of PDK3 dimer constitutes an integral part of the lipoyl-binding pocket in the N-terminal domain of the opposing subunit. The two swapped C-terminal tails promote conformational changes in active-site clefts of both PDK3 subunits, resulting in largely disordered ATP lids in the ADP-bound form. Our structural and biochemical data suggest that L2 binding stimulates PDK3 activity by disrupting the ATP lid, which otherwise traps ADP, to remove product inhibition exerted by this nucleotide. We hypothesize that this allosteric mechanism accounts, in part, for E2p-augmented PDK3 activity.     

Genetic reconstruction and characterization of the recombinant transacylase (E2b) component of bovine branched-chain alpha-keto acid dehydrogenase complex. Implication of histidine 391 as an active site residue

Genetically altered transacylase (E2b) proteins of the bovine branched-chain alpha-keto acid dehydrogenase complex were overexpressed in Escherichia coli and characterized. Deletion by PstI or Bal31 digestion of the amino-terminal region of the inner-core domain (residues 175-421) beyond residue 209 resulted in a complete loss of transacylase activity. The enzyme assay was carried out using [1-14C]isovaleryl-CoA and exogenous dihydrolipoamide as substrates. The removal of 4 residues (Thr-Ile-Pro-Ile) (residues 175-178) from the amino terminus of the inner-core domain significantly reduced the level of transacylase activity. The results establish that the segment between residues 175 and 209 is an integral part of the active site of E2b. The residue His-391 in the recombinant inner-core domain (E2b delta 167) was changed to Asn or Gln by site-directed mutagenesis. The wild-type and the two mutant inner-core domains were assembled into 24-mers as determined by gel filtration. However, both Asn and Gln mutations were accompanied by a complete loss of the enzymatic activity. Titration of the natural branched-chain alpha-keto dehydrogenase complex from pH 8 to 6 sharply reduced transacylase activity. The above data support the hypothesis that a conserved histidine residue in E2 acts as a general base for the transacylation reaction by analogy with E. coli chloramphenicol acetyltransferases.     

Human ferrochelatase: characterization of substrate-iron binding and proton-abstracting residues

The terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin IX to form protoheme, is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1). A number of highly conserved residues identified from the crystal structure of human ferrochelatase as being in the active site were examined by site-directed mutagenesis. The mutants Y123F, Y165F, Y191H, and R164L each had an increased K(m) for iron without an altered K(m) for porphyrin. The double mutant R164L/Y165F had a 6-fold increased K(m) for iron and a 10-fold decreased V(max). The double mutant Y123F/Y191F had low activity with an elevated K(m) for iron, and Y123F/Y165F had no measurable activity. The mutants H263A/C/N, D340N, E343Q, E343H, and E343K had no measurable enzyme activity, while E343D, E347Q, and H341C had decreased V(max)s without significant alteration of the K(m)s for either substrate. D340E had near-normal kinetic parameters, while D383A and H231A had increased K(m)s for iron. On the basis of these data and the crystal structure of human ferrochelatase, it is proposed that residues E343, H341, and D340 form a conduit from H263 in the active site to the protein exterior and function in proton extraction from the porphyrin macrocycle. The role of H263 as the porphyrin proton-accepting residue is central to catalysis since metalation only occurs in conjunction with proton abstraction. It is suggested that iron is transported from the exterior of the enzyme at D383/H231 via residues W227 and Y191 to the site of metalation at residues R164 and Y165 which are on the opposite side of the active site pocket from H263. This model should be general for mitochondrial membrane-associated eucaryotic ferrochelatases but may differ for bacterial ferrochelatases since the spatial orientation of the enzyme within prokaryotic cells may differ.     

Crystal structure of pyruvate dehydrogenase phosphatase 1 and its functional implications

Pyruvate dehydrogenase phosphatase 1 (PDP1) catalyzes dephosphorylation of pyruvate dehydrogenase (E1) in the mammalian pyruvate dehydrogenase complex (PDC), whose activity is regulated by the phosphorylation-dephosphorylation cycle by the corresponding protein kinases (PDHKs) and phosphatases. The activity of PDP1 is greatly enhanced through Ca2+ -dependent binding of the catalytic subunit (PDP1c) to the L2 (inner lipoyl) domain of dihydrolipoyl acetyltransferase (E2), which is also integrated in PDC. Here, we report the crystal structure of the rat PDP1c at 1.8 A resolution. The structure reveals that PDP1 belongs to the PPM family of protein serine/threonine phosphatases, which, in spite of a low level of sequence identity, share the structural core consisting of the central beta-sandwich flanked on both sides by loops and alpha-helices. Consistent with the previous studies, two well-fixed magnesium ions are coordinated by five active site residues and five water molecules in the PDP1c catalytic center. Structural analysis indicates that, while the central portion of the PDP1c molecule is highly conserved among the members of the PPM protein family, a number of structural insertions and deletions located at the periphery of PDP1c likely define its functional specificity towards the PDC. One notable feature of PDP1c is a long insertion (residues 98-151) forming a unique hydrophobic pocket on the surface that likely accommodates the lipoyl moiety of the E2 domain in a fashion similar to that of PDHKs. The cavity, however, appears more open than in PDHK, suggesting that its closure may be required to achieve tight, specific binding of the lipoic acid. We propose a mechanism in which the closure of the lipoic acid binding site is triggered by the formation of the intermolecular (PDP1c/L2) Ca2+ binding site in a manner reminiscent of the Ca2+ -induced closure of the regulatory domain of troponin C.     

Structural and functional insights into the molecular mechanisms responsible for the regulation of pyruvate dehydrogenase kinase 2

PDHK2 is a mitochondrial protein kinase that phosphorylates pyruvate dehydrogenase complex, thereby down-regulating the oxidation of pyruvate. Here, we present the crystal structure of PDHK2 bound to the inner lipoyl-bearing domain of dihydrolipoamide transacetylase (L2) determined with or without bound adenylyl imidodiphosphate. Both structures reveal a PDHK2 dimer complexed with two L2 domains. Comparison with apo-PDHK2 shows that L2 binding causes rearrangements in PDHK2 structure that affect the L2- and E1-binding sites. Significant differences are found between PDHK2 and PDHK3 with respect to the structure of their lipoyllysine-binding cavities, providing the first structural support to a number of studies showing that these isozymes are markedly different with respect to their affinity for the L2 domain. Both structures display a novel type II potassium-binding site located on the PDHK2 interface with the L2 domain. Binding of potassium ion at this site rigidifies the interface and appears to be critical in determining the strength of L2 binding. Evidence is also presented that potassium ions are indispensable for the cross-talk between the nucleotide- and L2-binding sites of PDHK2. The latter is believed to be essential for the movement of PDHK2 along the surface of the transacetylase scaffold.     

Sepsis alters pyruvate dehydrogenase kinase activity in skeletal muscle

Chronic sepsis promotes a stable increase in pyruvate dehydrogenase kinase (PDHK) activity in skeletal muscle. PDHK is found tightly bound to the pyruvate dehydrogenase (PDH) complex and as free kinase. We investigated the ability of sepsis to modify the activity of the PDHK intrinsic to the PDH and free PDHK. Sepsis was induced by the intraabdominal introduction of a fecal-agar pellet infected with E. coli and B. fragilis. Five days later, mitochondria were isolated from skeletal muscle and PDHK measured in mitochondrial extracts. Sepsis caused an approximate 2-fold stimulation of PDHK. The mitochondrial extracts from control and septic rats were fractionated by gel chromatography on Sephacryl S-300 to separate PDHK intrinsic to PDH complex and free PDHK. PDH complex eluted at void volume and was assayed for PDHK intrinsic to the complex. The activity of PDHK intrinsic to PDH complex was a significantly increased 3 fold during sepsis. Free PDHK activity eluted after the PDH complex and its activity was enhanced by 70% during sepsis. Incubation of PDHK intrinsic to PDH with dichloroactate, an uncompetitive inhibitor of PDHK, showed the PDHK from septic rats relatively less sensitive to inhibition than controls. These results indicate that sepsis induces stable changes in PDHK in skeletal muscle.     

Characterization of point mutations in patients with pyruvate dehydrogenase deficiency: role of methionine-181, proline-188, and arginine-349 in the alpha subunit.

Human pyruvate dehydrogenase (E1), a heterotetramer (alpha2beta2), is the first component of the pyruvate dehydrogenase complex (PDC). E1 catalyzes the thiamin pyrophosphate (TPP)-dependent decarboxylation of pyruvate and the reductive acetylation of the dihydrolipoamide acetyltransferase component. Site-directed mutagenesis was employed to recreate three point mutations in the alpha subunit identified in E1-deficient patients, M181V, R349H, and P188L (P188A mutant E1 was used because of the very low level of expression of P188L), to investigate the functional roles of these three amino acid residues. P188A mutant E1 was much less thermostable than the wild-type E1. The kcats of M181V and P188A mutant E1s determined in the PDC reaction were 38 and 24% of that of the wild-type enzyme, respectively. The apparent Km for TPP for M181V increased significantly (approx 250-fold when determined in the PDC assay), while the apparent Km for pyruvate increased by only about 3-fold. In contrast, P188A had similar Kms for the coenzyme and the substrate as the wild-type. Km values for R349H were not determined due to the extremely low activity of this mutant (1.2% of the wild-type E1-specific activity measured in the PDC assay). Wild-type E1 displayed a lag phase in the progress curve of the PDC reaction measured in the presence of low TPP concentrations (below 1 microM) only. All mutants had a lag phase that was not eliminated even at very high TPP concentrations, suggesting modifications in the conformation of the active site. Kinetic analysis indicated thiamin 2-thiothiazolone pyrophosphate (ThTTPP) to be an intermediate analog for wild-type human E1. M181V required a higher concentration of ThTTPP for inactivation than the wild-type and P188A E1s. The results of circular dichroism spectropolarimetry in the far UV region indicated that there were no major changes in the secondary structure of M181V, P188A, and R349H E1s. These mutant enzymes exhibited negative dichroic spectra at about 330 nm only in the presence of high TPP concentrations. This study suggests that arginine-349 is critical for E1's activity, methionine-181 is involved in the binding of TPP, and proline-188 is necessary for structural integrity of E1.     

Specific ion influences on self-association of pyruvate dehydrogenase kinase isoform 2 (PDHK2), binding of PDHK2 to the L2 lipoyl domain, and effects of the lipoyl group-binding site inhibitor, Nov3r

Association of the PDHK2 and GST-L2 (glutathione-S-transferase fused to the inner lipoyl domain (L2) of dihydrolipoyl acetyltransferase (E2)) dimers was enhanced by K+ with higher affinity K+ binding than occurs at the PDHK2 active site. Supporting a distinct K+ binding site, the NH4+ ion did not effectively replace K+ in aiding GST-L2 binding. With 50 mM K+, Pi enhanced interference by ADP, ATP, or pyruvate of PDHK2 binding to GST-L2. The inclusion of Pi with ADP or ATP plus pyruvate greatly hindered PDHK2 binding to GST-L2 and promoted PDHK2 forming a tetramer. Reciprocally, GST-L2 interference with ATP/ADP binding also required elevated K+ and was increased by Pi. Potent inhibition by Nov3r of E2-activated PDHK2 activity (IC50 of approximately 7.8 nM) required elevated K+ and Pi. Nov3r only modestly inhibited the low activity of PDHK2 without E2. By binding at the lipoyl group binding site, Nov3r prevented PDHK2 binding to E2 and GST-L2. Nov3r interfered with high-affinity binding of ADP and pyruvate via a Pi-dependent mechanism. Thus, GST-L2 binding to PDHK2 is supported by K+ binding at a site distinct from the active site. Pi makes major contributions to ligands interfering with PDHK2 binding to GST-L2, the conversion of PDHK2 dimer to a tetramer, and Nov3r (an acetyl-lipoate analog) interfering with binding of ADP and pyruvate. Pi is suggested to facilitate transmission within PDHK2 of the stimulatory signal of acetylation from the distal lipoyl-group binding site to the active site.     

Residues Arg114 and Arg337 are critical for the proper function of Escherichia coli gamma-glutamyltranspeptidase

To evaluate the importance of conserved Arg114 and Arg337 residues of Escherichia coli γ-glutamyltranspeptidase (EcGGT), Lys, Leu, or Asp-substituted mutants were constructed by site-directed mutagenesis. The wild-type and mutant enzymes were overexpressed in the recombinant E. coli M15 and purified by nickel-chelate chromatography to near homogeneity. With the exception of R114K, all the other mutants significantly lost GGT activity, confirming the importance of these two residues in EcGGT. Kinetic analysis of R114L, R114D, R337K, and R337L revealed a significant increase in K_m with a minor change in k_cat, leading to more than an 8-fold decrease in k_cat/K_m values. Mutations of Arg337 impaired the capability of autocatalytic processing of the enzyme. In vitro maturation experiments revealed that EcGGT precursor mutants, pro-R337K and pro-R337L, could precede a time-dependent autocatalytic process to generate the small and large subunits, while no autocatalytic processing was observed in pro-R337D. Computer modeling showed that the critical bonding distance of Gln390 O-Thr391 HG1 and Gln390 C-Thr391 OG1 are significantly increased in Arg337 replacements, implying that these distance changes might be responsible for the lack of enzyme maturation.     

The study of the bacteriophage T5 deoxynucleoside monophosphate kinase active site by site-directed mutagenesis

The amino acid residues essential for the enzymatic activity of bacteriophage T5 deoxyribonucleoside monophosphate kinase were determined using a computer model of the enzyme active site. By site-directed mutagenesis, cloning, and gene expression in E. coli, a series of proteins were obtained with single substitutions of the conserved active site amino acid residues—S13A, D16N, T17N, T17S, R130K, K131E, Q134A, G137A, T138A, W150F, W150A, D170N, R172I, and E176Q. After purification by ion exchange and affine chromatography electrophoretically homogeneous preparations were obtained. The study of the enzymatic activity with natural acceptors of the phosphoryl group (dAMP, dCMP, dGMP, and dTMP) demonstrated that the substitutions of charged amino acid residues of the NMP binding domain (R130, R172, D170, and E176) caused nearly complete loss of enzymatic properties. It was found that the presence of the OH-group at position 17 was also important for the catalytic activity. On the basis of the analysis of specific activity variations we assumed that arginine residues at positions 130 and 172 were involved in the binding to the donor γ-phosphoryl and acceptor α-phosphoryl groups, as well as the aspartic acid residue at position 16 of the ATP-binding site (P-loop), in the binding to some acceptors, first of all dTMP. Disproportional changes in enzymatic activities of partially active mutants, G137A, T138A, T17N, Q134A, S13A, and D16N, toward different substrates may indicate that different amino acid residues participate in the binding to various substrates.