Antagonistic substrate binding by a group II intron ribozyme.

In this study, the thermodynamic properties of substrate-ribozyme recognition were explored using a system derived from group II intron ai5gamma. Substrate recognition by group II intron ribozymes is of interest because any nucleic ac?id sequence can be targeted, the recognition sequence can be quite long (>/=13 bp), and reaction can proceed with a very high degree of sequence specificity. Group II introns target their substrates throug?h the formation of base-pairing interactions with two regions of the intron (EBS1 and EBS2), which are usually located far apart in the secondary structure. These structures pair with adjacent, corresponding sites (IBS1 and IBS2) on the substrate. In order to understand the relative energetic contribution of each base-pairing interaction (EBS1-IBS1 or EBS2-IBS2) to substrate binding energy, the free energy of each helix was measured. The individual helices were found to have base-pairing free energies similar to those calculated for regular RNA duplexes of the same sequence, suggesting that each recognition helix derives its binding energy from base-pairing interactions alone and that each helix can form independently. Most interestingly, it was found that the sum of the measured individual free energies (approximately 20 kcal/mol) was much higher than the known free energy for substrate binding (approximately 12 kcal/mol). This indicates that certain group II intron ribozymes can bind their substrates in an antagonistic fashion, paying a net energetic penalty upon binding the full-length substrate. This loss of binding energy is not due to weakening of individual helices, but appears to be linked to ribozyme conformational changes induced by substrate binding. This coupling between substrate binding and ribozyme conformational rearrangement may provide a mechanism for lowering overall substrate binding energy while retaining the full information content of 13 bp, thus resulting in a mechanism for ensuring sequence specificity.     

Role of hydroxyl group and R/S configuration of isostere in binding properties of HIV-1 protease inhibitors.

The crystal structure of the complex between human immunodeficiency virus type 1 (HIV-1) protease and a peptidomimetic inhibitor of ethyleneamine type has been refined to R factor of 0.178 with diffraction limit 2.5 A. The peptidomimetic inhibitor Boc-Phe-Psi[CH2CH2NH]-Phe-Glu-Phe-NH2 (denoted here as OE) contains the ethyleneamine replacement of the scissile peptide bond. The inhibitor lacks the hydroxyl group which is believed to mimic tetrahedral transition state of proteolytic reaction and thus is suspected to be necessary for good properties of peptidomimetic HIV-1 protease inhibitors. Despite the missing hydroxyl group the inhibition constant of OE is 1.53 nm and it remains in the nanomolar range also towards several available mutants of HIV-1 protease. The inhibitor was found in the active site of protease in an extended conformation with a unique hydrogen bond pattern different from hydroxyethylene and hydroxyethylamine inhibitors. The isostere nitrogen forms a hydrogen bond to one catalytic aspartate only. The other aspartate forms two weak hydrogen bridges to the ethylene group of the isostere. A comparison with other inhibitors of this series containing isostere hydroxyl group in R or S configuration shows different ways of accommodation of inhibitor in the active site. Special attention is devoted to intermolecular contacts between neighbouring dimers responsible for mutual protein adhesion and for a special conformation of Met46 and Phe53 side chains not expected for free protein in water solution.     

Inhibition and binding modes of low-molecular-weight inhibitors of porcine pancreatic alpha-amylase.

Inhibition of porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrase) [EC 3.2.1.1] with maltotriitol (G3OH) and 4-phenylimidazole was investigated by using maltohexaitol (G6OH) and p-nitrophenyl-alpha-D-maltoside (G2PNP) as substrates. When G6OH was the substrate, both G3OH and 4-phenylimidazole behaved as competitive inhibitors. On the other hand, when G2PNP was the substrate, G3OH behaved as a competitive inhibitor, whereas 4-phenylimidazole behaved as a non-competitive inhibitor. Further inhibition study in the presence of both G3OH and 4-phenylimidazole, with G6OH as the substrate, showed that the two inhibitors compete with each other for the active site of the enzyme. Based on a consideration of the productive (reactive) binding modes of G2PNP and G6OH, and a nonproductive (nonreactive) binding mode of G2PNP, it is suggested that the binding sites of the two inhibitors may be partially overlapping around the catalytic site of the enzyme and that the rest of the binding site of each inhibitor lies along the substrate binding cleft of the enzyme.     

Carbonic anhydrase inhibitors. N-cyanomethylsulfonamides--a new zinc binding group in the design of inhibitors targeting cytosolic and membrane-anchored isoforms

A series of N-cyanomethyl aromatic sulfonamides and bis-sulfonamides was prepared by reaction of arylsulfonyl halides with aminoacetonitrile. The obtained derivatives incorporated various aryl moieties, such as 4-halogeno/alkyl/aryl/nitro-substituted-phenyl, pentafluorophenyl or 2-naphthyl. Moderate inhibitory activity was detected for some compounds against the cytosolic human isoform II of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), hCA II, with inhibition constants of 90, 180 and 560n M for the 4-nitrophenyl-, 4-iodophenyl- and pentafluorophenyl-N-cyanomethylsulfonamides, respectively. Other derivatives acted as weak inhibitors of isoforms hCA I (KIs of 720 nM-45 microM), hCA II (KIs of 1000-9800 nM) and hCA IX (KIs of 900-10200 nM). Thus, the N-cyanomethylsulfonamide zinc binding group is less effective than the sulfonamide, sulfamate or sulfamide ones for the design of effective CA inhibitors.     

Brain pyridoxal kinase. Mobility of the substrate pyridoxal and binding of inhibitors to the nucleotide site.

Pyridoxal kinase has been purified 2000-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. The interactions of the substrate pyridoxal and the inhibitor N-dansyl-2-oxopyrrolidine (dansyl = 5-dimethylaminonaphthalene-1-sulfonyl) with the catalytic site were examined by means of fluorescence spectroscopy. The increase in emission anisotropy that follows the binding of pyridoxal to the kinase was used to determine the equilibrium dissociation constant. Pyridoxal kinase binds one molecule of substrate with a Kd = 11 microns at pH 6. The emission anisotropy spectrum of bound pyridoxal reveals that the substrate is not rigidly trapped by the protein matrix. N-Dansyl-2-oxopyrrolidine is a competitive inhibitor with respect to ATP at saturating concentrations of pyridoxal. It binds to the enzyme with a dissociation constant of 6 microns. N-Dansyl-2-oxopyrrolidine is immobilized by strong interactions with the enzyme, but it is displaced from the catalytic site by ATP. The results are consistent with the hypothesis that N-dansyl-2-oxopyrrolidine binds at the nucleotide binding site of pyridoxal kinase.     

Reactivity of the essential thiol group of lactate dehydrogenase and substrate binding

1. The preparation of a derivative of pig heart lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group is described. The derivative has unchanged s(20,w) and is catalytically inactive. 2. The rate of reaction of the essential thiol group is controlled by a system with a pK>9. 3. The essential thiol group is protected by NADH against reaction with maleimide. 4. Lactate dehydrogenase in which the essential thiol group has been converted into an S-sulpho group or alkylated with maleimide still binds one molecule of NADH/subunit but with a three- to four-fold diminished affinity. 5. The inhibited enzymes also bind one molecule of NAD(+)-sulphite complex/subunit but with affinity decreased 10(3)-10(4)-fold. 6. The inhibited enzymes fail to bind C(2) and C(3) molecules to give the ternary complexes enzyme-NAD(+)-pyruvate, enzyme-NADH-oxamate and enzyme-NADH-oxalate. The 1:1:1 stoicheiometry of the last-mentioned complex with the native enzyme was established by gel filtration. 7. Structures that account for these results are discussed.     

The tumour marker CA 195 in colorectal and pancreatic cancer.

The aim of this study was to measure the serum level of the tumour markers CA 195 and CEA in patients with either colorectal or pancreatic cancer both before and at serial intervals after operation. CA 195 and CEA were measured in 199 patients with colorectal cancer and 52 patients with pancreatic cancer. The median concentrations of CA 195 were 3.0 u/ml (interquartile range 3.0-4.5 u/ml) in patients with a Dukes' stage A lesion, 5.8 u/ml (3.0-18.2 u/ml) in patients with a Dukes' stage B lesion, 6.1 u/ml (3.0-24.7 u/ml) in patients with a Dukes' stage C and 23.8 u/ml (11.1-409.0 u/ml) in patients with metastatic disease (normal range 0-7 u/ml). The median levels of CEA were 2.6 ng/ml (1.7-3.3 ng/ml) for Dukes' stage A, 3.3 ng/ml (1.7-7.2 ng/ml) for Dukes' stage B, 3.7 ng/ml (2.2-7.9 ng/ml) for Dukes' stage C and 34.5 ng/ml (13.3-289.4 ng/ml) for metastatic disease. A rising level of CA 195 or CEA after operation suggested recurrence of the tumour. In none of these patients was the recurrence operable. In patients with pancreatic adenocarcinoma, the level of CA 195 was significantly higher in patients with metastatic disease but it did not discriminate between resectable and unresectable disease. The duration of survival correlated with the initial level of CA 195 (Rs = -0.66, p less than 0.001).     

Temperature-dependent cooperativity in donor-acceptor substrate binding to the human blood group glycosyltransferases

Affinities of the human blood group glycosyltransferases, alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) and alpha-(1-->3)-galactosyltransferase (GTB) for their common acceptor substrate alpha-l-Fucp-(1-->2)-beta-d-Galp-O(CH2)(7)CH3 (1), in the absence and presence of bound uridine 5'-diphosphate (UDP) and Mn2+ were determined using temperature-controlled electrospray ionization mass spectrometry. The presence of bound UDP and Mn(2+) in the donor binding site has a marked influence on the thermodynamic parameters for the association of 1 with GTA and GTB. Both the enthalpy and entropy of association (DeltaH(a), DeltaS(a)) decrease significantly. However, the free energy of association (DeltaG(a)) is unchanged at physiological temperature. The differences in the DeltaH(a) and DeltaS(a) values determined in the presence and absence of bound UDP are attributed to structural changes in the glycosyltransferases induced by the simultaneous binding of 1 and UDP.     

Role of the jelly-roll fold in substrate binding by 2-oxoglutarate oxygenases

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Highlights? Structural features of 2OG oxygenases involved in substrate recognition are analyzed. ? Crystallographic studies reveal the versatility of the jelly roll fold in substrate binding. ? Defined structural regions that interact with substrate(s) are biased by fold topology. ? The utility of the enzyme–substrate structures for engineering and selective inhibition are discussed.     

1H-n.m.r. studies on the existence of substrate binding sites in bovine pancreatic ribonuclease A

The reaction of ribonuclease A with either 6-chloropurine riboside 5'-monophosphate or the corresponding nucleoside yields one derivative, with the reagent covalently bound to the alpha-amino group of Lys-1, called derivative II and derivative E, respectively. We studied by means of 1H-n.m.r. at 270 MHz the interaction of these derivatives with different purine ligands. The pK values of His-12- and -119 were obtained and compared with those resulting from the interaction with ribonuclease A. The results showed that the interaction of derivative E with 3'AMP is similar to that described for RNase A as the pK2 of His-12 is increased while that of His-119 remains unaltered. However, derivative II presents some differences as it was found an enhancement of the pK2 values of both His-12 and His-119. Interaction of derivative II and derivative E with dApdA increases the pK2 of His-119, whereas a decrease is found when it interacts with ribonuclease A. These results suggest that the phosphate group and the nucleoside of both derivatives are located in regions of the enzyme where natural substrate analogues have secondary interactions and they can be interpreted as additional binding sites.     

Role of substrate binding forces in exchange-only transport systems: I. Transition-state theory

Summary An analysis of transition-state models for exchange-only transport shows that substrate binding forces, carrier conformational changes, and coupled substrate flow are interrelated. For a system to catalyze exchange but not net transport, addition of the substrate must convert the carrier from an immobile to a mobile form. The reduction in the energy barrier to movement is necessarily paid for out of the intrinsic binding energy between the substrate and the transport site, and is dependent on the formation of two different types of complex: a loose complex initially and a tight complex in the transition state in carrier movement. Hence the site should at first be incompletely organized for optimal binding but, following a conformational change, complementary to the substrate structure in the transition state. The conformational change, which may involve the whole protein, would be induced by cooperative interactions between the substrate and several groups within the site, involving a chelate effect. The tightness of coupling, i.e., the ratio of exchange to net transport, is directly proportional to the increased binding energy in the transition state, a relationship which allows the virtual substrate dissociation constant in the transition state to be calculated from experimental rate and half-saturation constants. Because the transition state is present in minute amount, strong bonding here does not enhance the substrate's affinity, and specificity may, therefore, be expressed in maximum exchange rates alone. However, where substrates largely convert the carrier to a transport intermediate whose mobility is the same with all substrates, specificity is also expressed in affinity. Hence the expression of substrate specificity provides evidence on the translocation mechanism.     

Adenosine di- and triphosphate transport in mitochondria. Role of the amidine region for substrate binding and transport

A variety of base-modified nucleotide analogues was prepared and characterized as their alpha-32P- or U-14C-labeled compounds. Carrier-linked nucleotide binding and carrier-catalyzed exchange across the inner membrane of rat liver mitochondria were measured by using an inhibitor (atractyloside) stop method. Kinetic data of carrier-specific bound analogues were evaluated from Dixon plots and indicate that these analogues are competitive inhibitors for mitochondrial [14C]ADP uptake. Km and Vmax values for carrier-mediated uptake of nucleotide analogues were calculated from Lineweaver-Burk plots. By means of the analogues, a systematic mapping of the essential chemical and steric interactions between the transporter protein and the heterocycle of its substrate in the course of the binding as well as transfer step was achieved. Prerequisites for carrier-specific binding (recognition) are (A) an anti- or syn-positioned beta-glycosyl-linked heterocycle, (B) a nitrogen ring atom in position 7 for syn-structured analogues, and (C) an electron-rich region at the N(1) position, i.e., a permanent dipole moment oriented toward N(1) for anti-structured analogues. Additional requirements for subsequent transport catalysis are (A) a non-fixed anti-positioned base moiety with a beta-glycosyl torsion angle of about -20 degrees, (B) a C(6)-positioned amino group, and (C) an unsubstituted C(2) atom. The complementary binding site at the carrier protein to the N(1)-C(6)(-NH2) amidine region is proposed to be represented by two juxtaposed and invariant bonding points, i.e., an asparagine or glutamine residue.     

Amino acid residues forming the active site of arylsulfatase A. Role in catalytic activity and substrate binding

Arylsulfatase A belongs to the sulfatase family whose members carry a Calpha-formylglycine that is post-translationally generated by oxidation of a conserved cysteine or serine residue. The formylglycine acts as an aldehyde hydrate with two geminal hydroxyls being involved in catalysis of sulfate ester cleavage. In arylsulfatase A and N-acetylgalactosamine 4-sulfatase this formylglycine was found to form the active site together with a divalent cation and a number of polar residues, tightly interconnected by a net of hydrogen bonds. Most of these putative active site residues are highly conserved among the eukaryotic and prokaryotic members of the sulfatase family. To analyze their function in binding and cleaving sulfate esters, we substituted a total of nine putative active site residues of human ASA by alanine (Asp29, Asp30, Asp281, Asn282, His125, His229, Lys123, Lys302, and Ser150). In addition the Mg2+-complexing residues (Asp29, Asp30, Asp281, and Asn282) were substituted conservatively by either asparagine or aspartate. In all mutants Vmax was decreased to 1-26% of wild type activity. The Km was more than 10-fold increased in K123A and K302A and up to 5-fold in the other mutants. In all mutants the pH optimum was increased from 4.5 by 0.2-0.8 units. These results indicate that each of the nine residues examined is critical for catalytic activity, Lys123 and Lys302 by binding the substrate and the others by direct (His125 and Asp281) or indirect participation in catalysis. The shift in the pH optimum is explained by two deprotonation steps that have been proposed for sulfate ester cleavage.     

Role of chemistry versus substrate binding in recruiting promiscuous enzyme functions

Two different scenarios for the recruitment of evolutionary starting points and their subsequent divergence to give new enzymes have been described. The coincidental, promiscuous starting activity may regard the same reaction chemistry on a new substrate (substrate ambiguity). Alternatively, substrate binding guides the recruitment of an enzyme whose reaction chemistry differs from that of the newly evolving one (catalytic promiscuity). While substrate ambiguity seems to underlie the divergence of most enzyme families, the relative levels of occurrence of these scenarios remain unknown. Screening the Escherichia coli proteome with a comparative series of xenobiotic substrates, we found that substrate ambiguity was, as anticipated, more frequent than reaction promiscuity. However, for at least one unnatural reaction (phosphonoesterase), a promiscuous enzyme was identified only when the substrate was decorated with the naturally abundant phosphate group. These findings support the prevailing hypothesis of chemistry-driven divergence but also suggest that recognition of familiar substrate motifs plays a role. In the absence of enzymes catalyzing the same chemistry, having a familiar, naturally occurring substrate motif (chemophore) such as phosphate may increase the likelihood of catalytic promiscuity. Chemophore anchoring may also find practical applications in identifying catalysts for unnatural reactions.     

Role of substrate functional groups in binding to nitric oxide synthase

The interactions between the heme CO ligand in the oxygenase domain of nitric oxide synthase and a set of substrate analogues were determined by measuring the resonance Raman spectra of the Fe-C-O vibrational modes. Substrates were selected that have variations in all the functional units: the guanidino group, the amino acid site and the number of methylene units connecting the two ends. In comparison to the substrate free form of the enzyme, Interactions of the analogues with the CO moiety caused the Fe-CO stretching and the Fe-C-O bending modes to shift in frequency due to the electrostatic environment. An unmodified guanidino group interacted with the CO in a similar fashion despite changes in the amino acid end. However, an unmodified amino acid end is required for catalysis owing to the H-bonding network involving the substrate, the heme and the pterin cofactor.     

Kinetic consequences of a slow substrate binding step in group transferases. Interpretation of product inhibition experiments.

The steady state kinetic properties of a simple model for an enzyme catalyzed group transfer reaction between two substrates have been calculated. One substrate is assumed to bind slowly and the other rapidly to the enzyme. Apparent substrate inhibition or substrate activation by the rapidly binding substrate may result if the slowly binding substrate binds at unequal rates to the free enzyme and to the complex between the enzyme and the rapidly binding substrate. Competitive inhibition by each product with respect to its structurally analogous substrate is to be expected if both substrates are in rapid equilibrium with their enzyme-substrate complexes. This product inhibition pattern, however, may also be observed when one substrate binds slowly. Noncompetitive inhibition with respect to the rapidly binding substrate by its structurally analogous product may result if the slowly binding substrate binds more slowly to the enzyme-product complex than to the free enzyme. Inhibition by substrate analogs which are not products should follow the same rules as inhibition by products. Thus substrate analog inhibition experiments are not particularly informative. The form of inhibition by "transition state analog" inhibitors should reveal which substrate binds slowly. There is no sharp conceptual distinction between ordered and random "kinetic mechanisms". I therefore suggest that the use of these concepts should be abandoned.