Linear Competitive Inhibitor Research Articles

Inhibition by glutathione derivatives of bovine liver glyoxalase II (hydroxyacylglutathione hydrolase) as a probe of the N- and S-sites for substrate binding

The nature of the binding determinants used in the interaction of glutathione-based derivatives and bovine liver glyoxalase II ( S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) has been investigated. Linear competitive inhibition was observed for S-blocked and S,N-blocked glutathiones with bovine liver glyoaxalase II (molecular weight 22 500 by sodium dodecyl sulphate polyacrylamide gel electrophoresis; pI = 7. 48 by analytical isoelectric focussing). There is a significant hydrophobic region on the enzyme to bind substituents around the sulphydryl-derived moiety of the substrate - a hydrophobic S-site. However, there is no evidence for binding of the N-site of the substrate (or inhibitor) to glyoxalase II. In contrast to glyoxalase I, there is no linkage between binding forces used at the S- and N-sites. Binding of S, N-dicarbobenzoxyglutathione is pH-dependent, showing dependence on an ionisation with p K app ≈ 7.2 (binding more tightly at higher pH), as is the k cat value (p K app) ≈ 7.8 for S- d- lactoylglutathione .

Kinetic and magnetic resonance studies of effects of genetic substitution of a Ca2+-liganding amino acid in staphylococcal nuclease.

The X-ray structure of staphylococcal nuclease suggests octahedral coordination of the essential Ca2+, with Asp-21, Asp-40, and Thr-41 of the enzyme providing three of the six ligands [Cotton, F. A., Hazen, E. E., Jr., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555]. The Asp-40 codon was mutated to Gly-40 on the gene that had been cloned into Escherichia coli, and the mutant (D40G) and wild-type enzymes were both purified from E. coli by a simple procedure. The D40G mutant forms a (5 +/- 2)-fold weaker binary complex with Ca2+ as found by kinetic analysis and by Ca2+ binding studies in competition with Mn2+, a linear competitive inhibitor. Similarly, as found by electron paramagnetic resonance (EPR), Mn2+ binds to the D40G mutant with a 3-fold greater KD than that found with the wild-type enzyme. These differences in KD are increased by saturation of staphylococcal nuclease with the DNA substrate such that KmCa is 10-fold greater and KIMn is 15-fold greater for the mutant than for the wild-type enzyme, although KMDNA is only 1.5-fold greater in the mutant. The six dissociation constants of the ternary enzyme-Mn2+-nucleotide complexes of 3',5'-pdTp and 5'-TMP were determined by EPR and by paramagnetic effects on 1/T1 of water protons, and the dissociation constants of the corresponding Ca2+ complexes were determined by competition with Mn2+. Only small differences between the mutant and wild-type enzymes are noted in K3, the dissociation constant of the nucleotides from their respective ternary complexes. 3',5'-pdTp raises the affinities of both wild-type and mutant enzymes for Mn2+ by factors of 47 and 31, respectively, while 5'-TMP raises the affinities of the enzymes for Mn2+ by smaller factors of 6.8 and 4.4, respectively. Conversely, Mn2+ raises the affinities of both wild-type and mutant enzymes for the nucleotides by 1-2 orders of magnitude. Analogous effects are observed in the ternary Ca2+ complexes. Dissociation constants of Ca2+ and Mn2+ from binary and ternary complexes, measured by direct binding studies, show reasonable agreement with those obtained by kinetic analysis. Structural differences in the ternary metal complexes of the D40G mutant are revealed by a 31-fold decrease in Vmax with Ca2+ and by 1.4-3.1-fold decreases in the enhancement of 1/T1 of water protons with Mn2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of a vinyl phosphonate analog of ATP as a rotationally constrained probe of the C5′O5′ torsion angle in ATP complexed to methionine adenosyl transferase

An analog of adenosine 5′-triphosphate (ATP) was synthesized in which the C4′C5′OP α system is replaced by a trans C4′CHCHP α system. In the form of 1:1 complexes with Mg, this analog and its counterpart with a C4′CH 2CH 2P α system were linear competitive inhibitors, with respect to MgATP, of the MAT-II (normal tissue) and MAT-T (hepatoma tissue) forms of rat ATP: l-methionine- S-adenosyltransferase (MAT); K m( ATP) K i values ranged from 0.4 to 2.4. 2′-Deoxy-ATP was a weak substrate, K m( ATP) K m = 0.035 , of MAT-II and a weak competitive inhibitor, K m( ATP) K i = 0.07 , of MAT-T. These findings, together with interactions of the MAT forms with other substrates and inhibitors, indicate that binding of ATP to these transferases is accompanied by little rotation about the C5′O5′ bond, and that C4′ and P α are in a trans-type relationship in enzyme-bound ATP.

Amino acid sequence of histone H1 at the ADP-ribose-accepting site and ADP-ribose X histone-H1 adduct as an inhibitor of cyclic-AMP-dependent phosphorylation.

The ADP-ribosylation site of histone H1 from calf thymus by purified hen liver nuclear ADP-ribosyltransferase was determined and effects of the ADP-ribose X histone-H1 adduct on cAMP-dependent phosphorylation of the histone H1 were investigated. ADP-ribosylated histone H1 was prepared by incubation of histone H1, 1 mM [adenylate-32P]NAD and the purified ADP-ribosyltransferase. N-Bromosuccinimide-directed bisection of ADP-ribosylated histone H1 showed that the NH2-terminal fragment (Mr = 6000) was modified and contained serine residue 38, the site of phosphorylation by cAMP-dependent protein kinase. Digestion of the NH2-terminal fragment with cathepsin D and trypsin, and purification of this fragment, using high-performance liquid chromatography, yielded a radiolabelled single peptide corresponding to residues 29-34 of histone H1, containing the arginine residue as the ADP-ribosylation site. These results indicate that ADP-ribosylation of histone H1 occurs at the arginine residue 34, sequenced at the NH2-terminal side of the phosphate-accepting serine residue 38. Phosphorylation of histone H1 from calf thymus by cAMP-dependent protein kinase was markedly reduced when histone H1 was ADP-ribosylated. Kinetic studies of phosphorylation revealed that ADP-ribosylated histone H1 was a linear competitive inhibitor of histone H1 and a linear non-competitive inhibitor of ATP.

Diagnosis of linear competitive inhibition

Diagnosis of linear competitive inhibition

Purification and properties of a carboxylesterase from germinated finger millet (Eleusine coracana Gaertn.)

A carboxylesterase (EC 3.1.1.1) was purified from germinated finger millet by ammonium sulphate fractionation, diethylaminoethyl-cellulose chromatography and Sephadex G-200 filtration. The homogeneity of the enzyme was established by Polyacrylamide gel electrophoresis, isoelectric focussing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme has a single polypeptide chain with a molecular weight of 70,000. The amino acid analysis of the purified enzyme revealed that it contained a greater number of neutral and acidic, compared to, basic amino acid residues. The isoelectric pH of the enzyme was found to be 5·1. Studies with different organophosphate and carbamate inhibitors showed that this enzyme was more sensitive to organophosphate inhibitors than carbamates. The rate constantski andl50 for different inhibitors were calculated. The product inhibition studies with this enzyme showed linear competitive inhibition with acetate and linear noncompetitive inhibition with 1-naphthol

Specificity of cGMP binding to a purified cGMP-stimulated phosphodiesterase from bovine adrenal tissue.

The binding of [3H]cGMP (guanosine 3',5'-monophosphate) to purified bovine adrenal cGMP-stimulated phosphodiesterase was measured by Millipore filtration on cellulose ester filter. [3H]cGMP-binding activity was enhanced when the assay was terminated in buffer containing 70% of saturated ammonium sulfate to dilute the enzyme and wash the filters. The cGMP-binding activity was co-purified with the phosphodiesterase activity. The binding of [3H]cGMP to purified enzyme was measured in the presence or absence of the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine. 1-Methyl-3-isobutylxanthine showed linear competitive inhibition with respect to cGMP as substrate in the phosphodiesterase reaction but stimulated the [3H]cGMP-binding activity in the binding assay. The stimulatory effect appeared not to be the result of preservation from [3H]cGMP hydrolysis; no cGMP phosphodiesterase activity has been measured under the cGMP-binding assay conditions, in the absence or presence of the inhibitor. Half-maximal stimulation by 1-methyl-3-isobutylxanthine occurred in the 5-7 microM concentration range. The specificity of binding of [3H]cGMP was investigated by adding increasing concentration of unlabeled analogs of cAMP (adenosine 3',5'-monophosphate) and cGMP. The binding of [3H]cGMP (50 nM) was displaced by unlabeled cGMP and cAMP with the following potency: 50% displacement was reached at the 0.1 microM cGMP range and only at a fiftyfold higher cAMP concentration. Our data with comparative series of analogs (e.g. 5'-amino-5'-deoxyguanosine 3',5'-monophosphate and 3'-amino-3'-deoxyguanosine 3',5'-monophosphate) showed that the potencies of stimulation of cAMP phosphodiesterase activity parallels displacement curves or [3H]cGMP binding to purified enzyme with no correlation with phosphodiesterase inhibition sequences. Those experiments suggest that the cGMP-binding activity is directly related to the non-catalytic (allosteric) cGMP-binding site.

Synthesis and characterization of the oxygen and desthio analogues of glutathione as dead-end inhibitors of glutathione S-transferase

The oxygen analogue, γ-L-Glu-L-SerGly (GOH) 1 1 Abbreviations used are: GOH, γ-L-glutamyl-L-serylglycine; GH, γ-L-glutamyl-L-alanylglycine; GSH, glutathione; SDS, sodium dodecyl sulfate; DCC, dicyclohexylcarbodiimide; TMS, tetramethylsilane; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; CDNB, 1-chloro-2,4-dinitrobenzene; MOPS, 3-(N-morpholino)-propanesulfonic acid; PIPES, piperazine N,N′-bis-(2-ethanesulfonic acid); t-BOC, tert -butyloxycarbonyl and desthio analogue, γ-L-Glu-L-AlaGly (GH) have been synthesized by a simple three step procedure involving active ester coupling of N-t-BOC-α-(4-nitrophenyl)-L-glutamate to L-SerGly and L-AlaGly, respectively. The two peptides are excellent dead-end inhibitors of isozymes 3-3 and 4-4 of rat liver glutathione S-transferase. At low fixed concentrations of 1-chloro-2,4-dinitrobenzene (CDNB) GOH and GH are linear competitive inhibitors of isozyme 3-3 vs glutathione with K I values of 13.0 and 116 μM, respectively. Both peptides are non-competitive (mixed-type) inhibitors vs CDNB when glutathione is the fixed substrate. Similar results are obtained with both peptides and isozyme 4-4. The results rule out ordered or ping-pong kinetic mechanisms where the electrophile adds first.

β(1–3)Glucan synthase activity of Neurospora crassa: Kinetic analysis of negative effectors

β(1–3)Glucan synthase activity of Neurospora crassa was inhibited by a number of compounds. Uridine nucleotides were linear competitive inhibitors of enzyme activity. Hill plots for the competitive inhibitors and for the substrate (UDP-glucose) resulted in straight lines with Hill numbers near unity suggesting a single substrate binding site. Tunicamycin, dolichol, or dolichol phosphate did not affect enzyme activity and a lipid-linked intermediate was not detected. Sorbose, gluconolactone, echinocandin B, and papulacandin B were uncompetitive inhibitors. Mixed inhibitor studies revealed that the binding of one uncompetitive inhibitor blocked completely the binding of each of the other uncompetitive inhibitors.

Mechanism-based inhibitors of dopamine beta-hydroxylase: inhibition by 2-bromo-3-(p-hydroxyphenyl)-1-propene.

The compound 2-bromo-3-(p-hydroxyphenyl)-1-propene (I) is a substrate of dopamine beta-hydroxylase from adrenal medulla, and the product has been identified by mass spectrometry as 2-bromo-3-hydroxy-3-(p-hydroxyphenyl)-1-propene (II). Compound I also inactivates dopamine beta-hydroxylase in a mechanism-based fashion. Thus, in acetate buffer at pH 5.0, inactivation by I exhibits saturation kinetics with a KD = 4.5 microM and kinact = 0.09 min-1. The inactivation is strictly dependent on O2 and a reducing agent (ascorbate or ferrocyanide) and is irreversible with no reactivation occurring upon prolonged dialysis or passage through a gel filtration column. The observed rate of inactivation at [I] = 4.5 mM (pH 5.5) increases from 0.045 to 0.17 min-1 when [O2] is increased from 0.25 to 1.2 mM. Norepinephrine affords competitive protection against inactivation of enzyme by I. In initial velocity experiments, I is a linear competitive inhibitor vs. tyramine. The log Kis vs. pH profile is flat while the log kinact vs. pH profile has an inflection corresponding to a group with a pKa of 5.7 +/- 0.1. These data demonstrate that an enzymic group in its protonated form is involved in the inactivation reaction and not in the binding of I to the enzyme. In addition, inactivation requires a catalytically competent enzyme, inasmuch as no inactivation occurs when Cu2+-free enzyme is incubated with I in the presence of ascorbate and O2.(ABSTRACT TRUNCATED AT 250 WORDS)

Chitin synthetase ofNeurospora crassa: Inhibition by nikkomycin, polyoxin B, and UDP

The inhibitory effects of nikkomycin, polyoxin B, and UDP were tested on particulate chitin synthetase activity (UDP-2-acetamido-2-deoxy-D-glucose: chitin-4-B-acetamidodeoxy-D-glucosyltransferase, E.C.2.4.1.16) fromNeurospora crassa. Two approaches were used: (a) inhibitors were tested for their individual effects on chitin synthetase activity; (b) paired combinations of inhibitors were examined to establish whether the compounds affected inhibition by binding at the same enzyme site. The first method showed that the three compounds are linear competitive inhibitors, i.e. each competes directly with the substrate for binding. Ki app values were: UDP, 0.8 mM; polyoxin B, 32 μM; and nikkomycin, 2 μM. The second method showed that the inhibitors compete with each other for binding; thus they bind at the same site. The pyrimidine nucleoside moiety of these inhibitors is an essential component for effective inhibition, but the potency of inhibition is critically dependent on the conformation of the side group attached to carbon 5 of the ribose sugar.

Chemical mechanism of saccharopine dehydrogenase (NAD +, l-lysine-forming) as deduced from initial rate pH studies

The variation of kinetic parameters with pH has been determined so as to gain insight into the chemical mechanism of the saccharopine dehydrogenase (NAD +, l-lysine-forming)-catalyzed reaction. In the direction of reductive condensation of lysine and α-ketoglutarate (reverse reaction), the V K profile for lysine shows a group with a p K of 6.3 must be unprotonated and a group with a p K of 8.0 must be protonated for activity. Similar p K's are obtained in the p K i profile for ornithine, which acts as a linear competitive inhibitor with respect to lysine. Temperature and solvent perturbation studies show that these groups are probably histidines. The V K profile for α-ketoglutarate reveals a single group with p K = 8.4 (probably lysine) that must be protonated. It is proposed that one of the histidines is involved in the binding of the ϵ-amino group of the substrate lysine and the positively charged lysine residue hydrogen bonds to the carbonyl oxygen of α-ketoglutarate. In the direction of saccharopine cleavage, the V K profile for saccharopine shows that two groups with p K values of 6.0 and 7.1, possibly a histidine and lysine, must be unprotonated for its reaction with the enzyme · NAD + complex. The log V-pH plots for the forward and reverse reactions both show sigmoidal curves. At low pH, the activity is lower for the forward reaction, and is higher for the reverse reaction. The ionization of a single group appears to be responsible for the change in activity. A tentative scheme for the chemical reaction is presented.

Reaction of (Na+ + K+)-dependent adenosine triphosphatase with inorganic phosphate. Regulation by Na+, K+, and nucleotides.

Effects of Na+, K+, and nucleotides on Mg2+-dependent phosphorylation of (Na+ + K+)-dependent adenosine triphosphatase by Pi were studied under equilibrium conditions. Na+ was a linear competitive inhibitor with respect to Mg2+ and a mixed inhibitor with respect to Pi. K+ was a partial inhibitor; it interacted with positive cooperativity and induced negative cooperativities in the interactions of Mg2+ and Pi with the enzyme. Adenyl-5'-yl (beta, gamma-methylene)diphosphonate, a nonhydrolyzable analog of ATP, interacted with negative cooperativity to inhibit phosphorylation in competition with Pi. ATP was also a competitive inhibitor. Na+ and K+ acted antagonistically, Na+ and nucleotides inhibited synergistically, and K+ and nucleotides were mutually exclusive. In the presence of ouabain, when nucleotides were excluded from the site inhibiting phosphorylation, a low affinity regulatory site for nucleotides became apparent, the occupation of which reduced the rate of dephosphorylation and the initial rate of phosphorylation of the enzyme without affecting the equilibrium constant of the reaction of Pi with the ouabain-complexed enzyme. The regulatory site was also detected in the absence of ouabain. The data suggest that catalytic and transport functions of the oligomeric enzyme may be regulated by homotropic and heterotropic site-site interactions, ligand-induced slow isomerizations, and distinct catalytic and regulatory sites for ATP.

5-Phosphoribosylpyrophosphate amidotransferase from soybean root nodules: Kinetic and regulatory properties

Most of the nitrogen transported from the nodules of nitrogen-fixing soybean plants is in the form of the ureides allantoin and allantoic acid. Recent work has shown that ureides are formed in the plant fraction of the nodule from de novo purine biosynthesis and purine oxidation. 5-Phosphoribosylpyrophosphate amidotransferase (PRAT), which catalyzes the first committed step of purine biosynthesis, has been purified 1500-fold from soybean root nodules. The enzyme had an apparent M r of 8 × 10 6, but this estimate may have been for an aggregation of several purine biosynthetic activities. PRAT showed a pH optimum of pH 8.0, and K m values were 18 and 0.4 m m for glutamine and 5-phosphoribosyl-1-pyrophosphate (PRPP), respectively. The reaction required Mg 2+, and PRPPMg 3− was shown to be the reactive molecular species of PRPP. Ammonia could replace glutamine as a substrate, and the V m with ammonia was twice that obtained when glutamine was the substrate. The initial-rate kinetics showed sequential addition of substrates to the enzyme. Product inhibition data was consistent with the order of product release being phosphoribosylamine, PP i, and glutamate. The enzyme was subject to regulation by end products of the purine biosynthetic pathway. IMP and GMP inhibited competitively with PRPP and promoted cooperativity in the binding of this substrate; there was no cooperativity in the binding of IMP to the enzyme. XMP was a linear competitive inhibitor with PRPP. The results are discussed in terms of the key regulatory point occupied by PRAT in the pathway of ureide biogenesis.

Soluble aminopeptidases from cytosol and luminal contents of Rhynchosciara americana midgut caeca : Properties and phenanthroline inhibition

Rhynchosciara americana midgut caecal cells contain two major cytosolic aminopeptidases which are resolved by electrophoresis but which have the same M r value (115,700), as determined by gradient ultracentrifugation, and have pI values of 8.7 and 7.8. Electrophoretic migration of the two aminopeptidases in polyacrylamide gels of different concentration suggests they differ only in net charge. Thermal inactivation of both cytosolic aminopeptidases follow apparent first-order kinetics with identical half-lives. The two cytosolic aminopeptidases have the same K m values when either leucine-p- nitroanilide or arginine-p- nitroanilide are substrates. Midgut luminal fluid displays two major aminopeptidases resolved by electrophoresis which have the same properties as the two cytosolic aminopeptidases. The cytosolic aminopeptidases purified by electrophoresis have the same pH optimum of 8.0 and Tris ( K i 107 mM) and 1,10-phenanthroline ( K i 14 μM) both act as simple linear competitive inhibitors. The enzymes are true aminopeptidases with a broad specificity towards aminoacyl-β-naphthylamides and are more active on tetra and tripeptides than on dipeptides. The data support the assumption that the cytosolic aminopeptidases from caecal cells, which are similar to those in luminal fluid, are enzymes en route to their being secreted and that they differ only in net charge. Furthermore, the properties of the aminopeptidases are in accordance to their proposed role of oligopeptide digestion in the ectoperitrophic fluid.

Irreversible inhibition of phosphotransacetylase by S-dimethylarsino-CoA

S-Dimethylarsino-CoA was synthesized by acylation of CoA with dimethylchloroarsine. The new analogue of acetyl-CoA was tested as an active-site-directed irreversible inhibitor of phosphotrasacetylase (EC. 2.3.1.8), carnitine acetyltransferase (EC 2.3.1.7) and citrate synthase (EC 4.1.3.7). Irreversible inhibition was observed only with phosphotransacetylase, which was derivatized via a simple bimolecular process ( k 2 = 197 ± 15 min −1· M −1 ). Acetyl-CoA provided complete substrate protection against the inactivation, while phosphate (a substrate) and desulfo-CoA (a competitive inhibitor) provided a partial protection. The inactivation was not reversed by dithiothreitol. The new reagent was a linear competitive inhibitor versus acetyl-CoA with both carnitine acetyltransferase ( K i = 41 μM) and citrate synthase ( K i = 20 μM). Chemical studies showed that S-dimethylarsino-CoA reacts with the thiol of N α -acetylcysteine but not with the side-chain functional groups of histidine and lysine. The nature of the chemical modification of cysteine was determined by investigating a model system. Thus the chemical reaction between the thioarsenite linkage of S-dimethylarsinobenzylmercaptan and the thiol of cysteine was shown to involve transesterification of the dimethylarsino group.

Characterization of phosphodiesterase catalytic sites by means of cyclic nucleotide derivatives.

Cyclic nucleotide derivatives have been used as a tool to characterize distinct catalytic sites on phosphodiesterase enzyme forms: the cGMP-stimulated enzyme from rat liver and the calmodulin-sensitive enzyme from rat or bovine brain. Under appropriate assay conditions, the analogues showed linear competitive inhibition with respect to cAMP (adenosine 3',5'-monophosphate) as substrate. The inhibition sequence of the fully activated cGMP-stimulated phosphodiesterase was identical to the inhibition sequence of the desensitized enzyme, i.e. the enzyme which has lost its ability to be stimulated by cGMP. The inhibition pattern could, therefore, not be attributed to competition with cGMP at an allosteric-activating site. Also, the inhibition sequence of the calmodulin-sensitive phosphodiesterase was maintained whether activity was basal or fully stimulated by calmodulin. When cAMP and cGMP, with identical chemical ligands substituted at the same position, were compared as inhibitors of the calmodulin-sensitive phosphodiesterase, the cGMP analogues were always the more potent suggesting that, for that enzyme, the catalytic site was sensitive to a guanine-type cyclic nucleotide structure. Comparing the two phosphodiesterases, it was possible to establish both similar and specific inhibitor potencies of cyclic nucleotide derivatives. In particular, the two enzymes exhibited large differences in analogue specificity modified at C-6, 6-chloropurine 3',5'-monophosphate or purine 3',5'-monophosphate.

PH dependence of the inhibition of yeast glyoxalase I by porphyrins

A number of porphyrin derivatives have been found to inhibit yeast glyoxalse I (EC 4.4.1.5) at 25°C, including haemin, protporphyrin IX, coproporphyrin III, haematoporphyrin, deuteroporphyrin as well as meso-(tetrasubstituted)porphines. Bilirubin and chlorophyllin were also inhibitory, but not cobalamin, adipic, pimelic or suberic acids. Whilst the K i value for linear competitive inhibition by meso-tetra(4-methylpyridyl)porphine was pH-dependent, analogous K i values for meso-tetra(4-carboxyphenyl)- and meso-tetra(4-sulphonatophenyl)porphines followed the Henderson-Hasselbalch equation with p I app values of 7.10 and 6.50, respectively. Protoporphyrin showed similar behaviour (p k app 7.06) with a deviation at lower pH. The haemin pH profile for K i showed a maximum at approx. pH 6.5. The redox reaction between haemin and glutathione did not interfere in the inhibition studies. The K i value for S-( p-bromobenzyl)glutatione was pH-independent. A detailed analysis of porphyrin binding modes was undertaken.

The interaction of fructose 2,6-bisphosphate with an allosteric site of rat liver fructose 1,6-bisphosphatase

Rat liver fructose 1,6-bisphosphatase can be protected against partial inactivation by N-ethylmaleimide by low concentrations of fructose 2,6-bisphosphate or high concentrations of fructose 1,6-bisphosphate. The partially inactivated enzyme has a much reduced sensitivity to high substrate inhibition and has lost the sigmoid component of the inhibition by fructose 2,6-bisphosphate; this compound is a simple linear competitive inhibitor of the modified enzyme. The results suggest that fructose 2,6-bisphosphate can bind to the enzyme at two distinct sites, the catalytic site and an allosteric site. High levels of fructose 1,6-bisphosphate probably inhibit by binding to the allosteric site.

Antiherpetic activity and mechanism of action of 9-(4-hydroxybutyl)guanine

9-(4-Hydroxybutyl)guanine was synthesized and tested for antiherpes activity. In cell cultures, different strains of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) were inhibited by 50% at 2–14 μM of 9-(4-hydroxybutyl)guanine, while a HSV-1 mutant lacking thymidine kinase (HSV-1 TK -) was resistant. Linear competitive inhibition of purified HSV-1-induced thymidine kinase (TK) with thymidine as a variable substrate was observed for 9-(4-hydroxybutyl)guanine with an apparent K i value of 2.06 μM while the corresponding K i value for the cellular TK was > 250 μM. By using high performance liquid chromatography, the formation of 9-(4-hydroxybutyl)guanine monophosphate by HSV-1 TK was measured and the rate of product formation was found to be about 10% of that found by using thymidine as a substrate. A selective inhibition of HSV-1 DNA synthesis by 9-(4-hydroxybutyl)guanine was observed in infected Vero cells. 9-(4-Hydroxybutyl)guanine had a low cellular toxicity. A weak therapeutic effect on herpes keratitis in rabbits was observed whereas cutaneous HSV-1 infection in guinea pigs and systemic HSV-2 infection in mice were not affected by this compound.