Noncatalytic Sites Research Articles

Fructose‐1,6‐bisphosphate aldolase (class II) is the primary site of nickel toxicity in Escherichia coli

Nickel is toxic to all forms of life, but the mechanisms of cell damage are unknown. Indeed, environmentally relevant nickel levels (8 µM) inhibit wild-type Escherichia coli growth on glucose minimal medium. The same concentration of nickel also inhibits growth on fructose, but not succinate, lactate or glycerol; these results suggest that fructose-1,6-bisphosphate aldolase (FbaA) is a target of nickel toxicity. Cells stressed by 8 µM Ni(II) for 20 min lost 75% of their FbaA activity, demonstrating that FbaA is inactivated during nickel stress. Furthermore, overexpression of fbaA restored growth of an rcnA mutant in glucose minimal medium supplemented with 4 µM Ni(II), thus confirming that FbaA is a primary target of nickel toxicity. This class II aldolase has an active site zinc and a non-catalytic zinc nearby. Purified FbaA lost 80 % of its activity within 2 min when challenged with 8 µM Ni(II). Nickel-challenged FbaA lost 0.8 zinc and gained 0.8 nickel per inactivated monomer. FbaA mutants (D144A and E174A) affecting the non-catalytic zinc were resistant to nickel inhibition. These results define the primary site of nickel toxicity in E. coli as the class II aldolase FbaA through binding to the non-catalytic zinc site.

Gilead Licenses Boehringer Ingelheim’s NCINI Inhibitors for HIV

Boehringer Ingelheim has granted Gilead Sciences exclusive worldwide rights for the research, development and commercialisation of its novel non-catalytic site integrase inhibitors (NCINIs) for HIV, including the lead compound BI-224436, which has completed a Phase Ia dose escalation study in healthy volunteers. In return, Boehringer Ingelheim will receive an upfront payment, potential development, regulatory and commercial milestone payments as well as royalties. The licensed compounds are complementary to Gilead’s internal discovery programmes.

Non-catalytic glycogen-binding site and its functional role in glycogen synthase

Non-catalytic glycogen-binding site and its functional role in glycogen synthase

Small-Molecule Anticancer Compounds Selectively Target the Hemopexin Domain of Matrix Metalloproteinase-9

Lack of target specificity by existing matrix metalloproteinase (MMP) inhibitors has hindered antimetastatic cancer drug discovery. Inhibitors that bind to noncatalytic sites of MMPs and disrupt protease signaling function have the potential to be more specific and selective. In this work, compounds that target the hemopexin (PEX) domain of MMP-9 were identified using an in silico docking approach and evaluated using biochemical and biological approaches. Two of the selected compounds interfere with MMP-9-mediated cancer cell migration and proliferation in cells expressing exogenous or endogenous MMP-9. Furthermore, these inhibitors do not modulate MMP-9 catalytic activity. The lead compound, N-[4-(difluoromethoxy)phenyl]-2-[(4-oxo-6-propyl-1H-pyrimidin-2-yl)sulfanyl]-acetamide, specifically binds to the PEX domain of MMP-9, but not other MMPs. This interaction between the compound and the PEX domain results in the abrogation of MMP-9 homodimerization and leads to blockage of a downstream signaling pathway required for MMP-9-mediated cell migration. In a tumor xenograft model, this pyrimidinone retarded MDA-MB-435 tumor growth and inhibited lung metastasis. Thus, we have shown for the first time that a novel small-molecule interacts specifically with the PEX domain of MMP-9 and inhibits tumor growth and metastasis by reducing cell migration and proliferation.

An Allosteric Inhibitor of the Human Cdc34 Ubiquitin-Conjugating Enzyme

In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.

Processivity and Subcellular Localization of Glycogen Synthase Depend on a Non-catalytic High Affinity Glycogen-binding Site

Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synthases has a large impact when glycogen is the acceptor substrate. Instead, the catalytic efficiency remains essentially unchanged when small oligosaccharides are used as substrates. Mutants of the human muscle enzyme with reduced affinity for glycogen also show an altered intracellular distribution and a marked decrease in their capacity to drive glycogen accumulation in vivo. The presence of a high affinity glycogen-binding site away from the active center explains not only the long-recognized strong binding of glycogen synthase to glycogen but also the processivity and the intracellular localization of the enzyme. These observations demonstrate that the glycogen-binding site is a critical regulatory element responsible for the in vivo catalytic efficiency of GS.

Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6, initiates a large conformational change involved in its deactivation

Retinal photoreceptor phosphodiesterase (PDE6), a key enzyme for phototransduction, consists of a catalytic subunit complex (Pαβ) and two inhibitory subunits (Pγs). Pαβ has two noncatalytic cGMP-binding sites. Here, using bovine PDE preparations, we show the role of these cGMP-binding sites in PDE regulation. Pαβγγ and its transducin-activated form, Pαβγ, contain two and one cGMP, respectively. Only Pαβγ shows [(3)H]cGMP binding with a K(d) ∼ 50 nM and Pγ inhibits the [(3)H]cGMP binding. Binding of cGMP to Pαβγ is suppressed during its formation, implying that cGMP binding is not involved in Pαβγγ activation. Once bound to Pαβγ, [(3)H]cGMP is not dissociated even in the presence of a 1000-fold excess of unlabeled cGMP, binding of cGMP changes the apparent Stokes' radius of Pαβγ, and the amount of [(3)H]cGMP-bound Pαβγ trapped by a filter is spontaneously increased during its incubation. These results suggest that Pαβγ slowly changes its conformation after cGMP binding, i.e. after formation of Pαβγ containing two cGMPs. Binding of Pγ greatly shortens the time to detect the increase in the filter-trapped level of [(3)H]cGMP-bound Pαβγ, but alters neither the level nor its Stokes' radius. These results suggest that Pγ accelerates the conformational change, but does not add another change. These observations are consistent with the view that Pαβγ changes its conformation during its deactivation and that the binding of cGMP and Pγ is crucial for this change. These observations also imply that Pαβγγ changes its conformation during its activation and that release of Pγ and cGMP is essential for this change.

Discovery of Orally Active, Potent, and Selective Benzotriazole‐Based PTP1B Inhibitors

Discovery of Orally Active, Potent, and Selective Benzotriazole‐Based PTP1B Inhibitors

Templated nucleoside triphosphate binding to a noncatalytic site on RNA polymerase regulates transcription

The regulation of RNA synthesis by RNA polymerase (RNAP) is essential for proper gene expression. Crystal structures of RNAP reveal two channels: the main channel that contains the downstream DNA and a secondary channel that leads directly to the catalytic site. Although nucleoside triphosphates (NTPs) have been seen only in the catalytic site and the secondary channel in these structures, several models of transcription elongation, based on biochemical studies, propose that template-dependent binding of NTPs in the main channel regulates RNA synthesis. These models, however, remain controversial. We used transient state kinetics and a mutant of RNAP to investigate the role of the main channel in regulating nucleotide incorporation. Our data indicate that a NTP specific for the i + 2 template position can bind to a noncatalytic site and increase the rate of RNA synthesis and that the NTP bound to this site can be shuttled directly into the catalytic site. We also identify fork loop 2, which lies across from the downstream DNA, as a functional component of this site. Taken together, our data support the existence of a noncatalytic template-specific NTP binding site in the main channel that is involved in the regulation of nucleotide incorporation. NTP binding to this site could promote high-fidelity processive synthesis under a variety of environmental conditions and allow DNA sequence-mediated regulatory signals to be communicated to the active site.

Templated nucleoside triphosphate binding to a noncatalytic site on RNA polymerase regulates transcription

Templated nucleoside triphosphate binding to a noncatalytic site on RNA polymerase regulates transcription

A geometry-based generic predictor for catalytic and allosteric sites

An important aspect of understanding protein allostery, and of artificial effector design, is the characterization and prediction of substrate- and effector-binding sites. To find binding sites in allosteric enzymes, many of which are oligomeric with allosteric sites at domain interfaces, we devise a local centrality measure for residue interaction graphs, which behaves well for both small/monomeric and large/multimeric proteins. The measure is purely structure based and has a clear geometrical interpretation and no free parameters. It is not biased towards typically catalytic residues, a property that is crucial when looking for non-catalytic effector sites, which are potent drug targets.

Regulation of matrix metalloproteinase activity in health and disease

The activity of matrix metalloproteinases (MMPs) is regulated at several levels, including enzyme activation, inhibition, complex formation and compartmentalization. Regulation at the transcriptional level is also important, although this is not a subject of the present minireview. Most MMPs are secreted and have their function in the extracellular environment. This is also the case for the membrane-type MMPs (MT-MMPs). MMPs are also found inside cells, both in the nucleus, cytosol and organelles. The role of intracellular located MMPs is still poorly understood, although recent studies have unraveled some of their functions. The localization, activation and activity of MMPs are regulated by their interactions with other proteins, proteoglycan core proteins and/or their glycosaminoglycan chains, as well as other molecules. Complexes formed between MMPs and various molecules may also include interactions with noncatalytic sites. Such exosites are regions involved in substrate processing, localized outside the active site, and are potential binding sites of specific MMP inhibitors. Knowledge about regulation of MMP activity is essential for understanding various physiological processes and pathogenesis of diseases, as well as for the development of new MMP targeting drugs.

Nucleotide binding to noncatalytic sites is essential for ATP-dependent stimulation and ADP-dependent inactivation of the chloroplast ATP synthase.

Light-dependent binding of labeled ADP and ATP to noncatalytic sites of chloroplast ATP synthase and the effect of light-exposed thylakoid membrane preincubation with ADP or ATP on ATPase activity were studied. ADP binding during the preincubation was shown to inactivate the chloroplast ATPase, whereas ATP binding caused its activation. The rate and equilibrium constants of ATPase inactivation and activation were close to those of ADP and ATP binding to a noncatalytic site, with K (d) values of 38 and 33μM, respectively. It is suggested that ADP- or ATP-binding to one of the noncatalytic sites affects the ATPase activity of chloroplast ATP synthase through a mechanism that modulates tightness of ADP binding to a catalytic site.

Effect of Noncompetitive Proteasome Inhibition on Bortezomib Resistance

Bortezomib and the other proteasome inhibitors that are currently under clinical investigation bind to the catalytic sites of proteasomes and are competitive inhibitors. We hypothesized that proteasome inhibitors that act through a noncompetitive mechanism might overcome some forms of bortezomib resistance. 5-amino-8-hydroxyquinoline (5AHQ) was identified through a screen of a 27-compound chemical library based on the quinoline pharmacophore to identify proteasome inhibitors. Inhibition of proteasome activity by 5AHQ was tested by measuring 7-amino-4-methylcoumarin (AMC) release from the proteasome substrate Suc-LLVY-AMC in intact human and mouse leukemia and myeloma cells and in tumor cell protein extracts. Cytotoxicity was assessed in 5AHQ-treated cell lines and primary cells from myeloma and leukemia patients using AlamarBlue fluorescence and MTS assays, trypan blue staining, and annexin V staining. 5AHQ-proteasome interaction was assessed by nuclear magnetic resonance. 5AHQ efficacy was evaluated in three leukemia xenograft mouse models (9-10 mice per group per model). All statistical tests were two-sided. 5AHQ inhibited the proteasome when added to cell extracts and intact cells (the mean concentration inhibiting 50% [IC(50)] of AMC release in intact cells ranged from 0.57 to 5.03 microM), induced cell death in intact cells from leukemia and myeloma cell lines (mean IC(50) values for cell growth ranged from 0.94 to 3.85 microM), and preferentially induced cell death in primary myeloma and leukemia cells compared with normal hematopoietic cells. 5AHQ was equally cytotoxic to human myelomonocytic THP1 cells and to THP1/BTZ500 cells, which are 237-fold more resistant to bortezomib than wild-type THP1 cells because of their overexpression and mutation of the bortezomib-binding beta5 proteasome subunit (mean IC(50) for cell death in the absence of bortezomib, wild-type THP1: 3.7 microM, 95% confidence interval = 3.4 to 4.0 microM; THP1/BTZ500: 6.6 microM, 95% confidence interval = 5.9 to 7.5 microM). 5AHQ interacted with the alpha subunits of the 20S proteasome at noncatalytic sites. Orally administered 5AHQ inhibited tumor growth in all three mouse models of leukemia without overt toxicity (eg, OCI-AML2 model, median tumor weight [interquartile range], 5AHQ vs control: 95.7 mg [61.4-163.5 mg] vs 247.2 mg [189.4-296.2 mg], P = .002). 5AHQ is a noncompetitive proteasome inhibitor that is cytotoxic to myeloma and leukemia cells in vitro and inhibits xenograft tumor growth in vivo. 5AHQ can overcome some forms of bortezomib resistance in vitro.

Evaluation of Inhibitory Actions of Flavonols and Related Substances on Lysophospholipase D Activity of Serum Autotaxin by a Convenient Assay Using a Chromogenic Substrate

Overproduction of lysophosphatidic acid (LPA) by lysophospholipase D/autotaxin (lysoPLD/ATX) is postulated to be involved in the promotion of cancer and atherosclerosis. A lysoPLD inhibitor may be utilized to ameliorate the LPA-related pathological conditions. In this study, a new assay was devised to quantify p-nitrophenol from hydrolysis of chromogenic substrate by serum lysoPLD without tedious lipid extraction procedures. Flavonols, phenolic acids, free fatty acids, and N-acyltyrosines inhibited lysoPLD activity in a micromolar range. They were classified into competitive, noncompetitive, or mixed type inhibitors. The results show that the low hydrophobicity of an inhibitor is a critical factor in its preference for the binding to a noncatalytic binding site over a catalytic binding site. Considering its reported bioavailability and the low dependency of its inhibitory activity on serum dilution, flavonol is likely to be a more effective lysoPLD inhibitor in human blood circulation in vivo than the other inhibitors including LPA.

Structure‐function studies of the regulatory bicarbonate binding in H. influenzae carbonic anhydrase: a functional reversion of allostery using multiple site mutations

Type II β‐carbonic anhydrases are metalloenzymes that catalyze the conversion of carbon dioxide to bicarbonate using a characteristic closed four‐side chain coordination sphere for the active site Zn ion. Non‐catalytic bicarbonate binding sites have been discovered in some type II β‐CAs 8 Å away from the active site. A network of seven hydrogen bonds holds the inhibitory bicarbonate in place, and kinetic studies confirm cooperative inhibition by bicarbonate at sufficient levels. A comparison of bicarbonate‐bound HICA with type I β‐CAs lacking the non‐catalytic bicarbonate binding site reveals a number of principal interactions that might be responsible for the allosteric regulation of HICA. W39 provides a direct, stabilizing hydrogen bond to the inhibitory bicarbonate, a one‐residue shift of proline 48 changes an influential backbone orientation of the 44–48 loop, and a G41 residue makes additional room in the allosteric binding site compared to a conserved Ala in non‐allosteric β‐CAs. Individual site mutants of W39 have revealed predictable decreases in cooperativity consistent with the loss of a stabilizing hydrogen bond to the bicarbonate. Structure function studies of compound site mutants, however, abrogate the binding of inhibitory bicarbonate in the crystalline state even at high concentrations.This work is supported by NSF grant CHE‐0819686 and MCB‐0741396.

Role of short conserved segments of α- and β-subunits that link F1-ATPase catalytic and noncatalytic sites

An analysis of amino acid sequences and 3D structures of chloroplast, mitochondrial, and bacterial F(1)-ATPases revealed that in their alpha- and beta-chains there are short highly conserved segments linking in pairs the catalytic and noncatalytic sites. The analysis was based on the reported effect of directed mutagenesis of amino acids forming these segments on catalytic properties of the F(1)-ATPases. It is proposed that one of these segments is responsible for transduction of a conformation signal from the noncatalytic to catalytic site upon ADP-for-ATP substitution at the noncatalytic site. At the catalytic site, this signal changes position of the terminal amino acid residue with respect to the adenine part of the molecule and results in a lower tightness of MgADP binding and its dissociation followed by enzyme activation. Mutagenesis of amino acids comprised by the two other segments was shown to produce an effect on the rate of cooperative catalysis, whereas the rate of single-site catalysis remained unaffected. This suggests that these segments are responsible for the cooperative mode of enzyme functioning.

Subunit Movements in Single Membrane-bound H+-ATP Synthases from Chloroplasts during ATP Synthesis

Subunit movements within the H(+)-ATP synthase from chloroplasts (CF(0)F(1)) are investigated during ATP synthesis. The gamma-subunit (gammaCys-322) is covalently labeled with a fluorescence donor (ATTO532). A fluorescence acceptor (adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP)-ATTO665) is noncovalently bound to a noncatalytic site at one alpha-subunit. The labeled CF(0)F(1) is integrated into liposomes, and a transmembrane pH difference is generated by an acid base transition. Single-pair fluorescence resonance energy transfer is measured in freely diffusing proteoliposomes with a confocal two-channel microscope. The fluorescence time traces reveal a repetitive three-step rotation of the gamma-subunit relative to the alpha-subunit during ATP synthesis. Some traces show splitting into sublevels with fluctuations between the sublevels. During catalysis the central stalk interacts, with equal probability, with each alphabeta-pair. Without catalysis the central stalk interacts with only one specific alphabeta-pair, and no stepping between FRET levels is observed. Two inactive states of the enzyme are identified: one in the presence of AMPPNP and one in the presence of ADP.

Cyclic Nucleotide Binding GAF Domains from Phosphodiesterases: Structural and Mechanistic Insights

GAF domains regulate the catalytic activity of certain vertebrate cyclic nucleotide phosphodiesterases (PDEs) by allosteric, noncatalytic binding of cyclic nucleotides. GAF domains arranged in tandem are found in PDE2, -5, -6, -10, and -11, all of which regulate the cellular concentrations of the second messengers cAMP and/or cGMP. Nucleotide binding to GAF domains affects the overall conformation and the catalytic activity of full-length PDEs. The cyclic nucleotide-bound GAF domains from PDE2, -5, -6, and -10 all adopt a conserved fold but show subtle differences within the binding pocket architecture that account for a large range of nucleotide affinities and selectivity. NMR data and details from the structure of full-length nucleotide-free PDE2A reveal the dynamic nature and magnitude of the conformational change that accompanies nucleotide binding. The discussed GAF domain structures further reveal differences in dimerization properties and highlight the structural diversity within GAF domain-containing PDEs.

FoF1-ATPase activity regulated by external links on β subunits

FoF1-ATPase activity is regulated by external links on β subunits with different molecular weight. It is inhibited when anti-β subunit antibody, streptavidin and H9 antibody link on the β subunits successively, but is activated when virus was binded. Western blotting indicated that the employed anti-β antibody target was on the non-catalytic site of the β subunit. Furthermore, an ESR study of spin-labeled ATP (SL-ATP) showed that the affinity of ATP to the holoenzyme increases with increasing external links on the β subunits. This simple regulation method may have great potential in the design of rapid, free labeled, sensitive and selective biosensors.