Anomeric specificity of D-xylose isomerase.

Crystal structures of complexes of D-xylose isomerase with deoxysugars have been determined. Deoxynojirimycin is a structural analogue of alpha-pyranose and mimics the binding of these aldose substrates. The structure of this complex supports the hypothesis that an imidazole group catalyzes ring opening of the pyranose. The steric restrictions in the active site of the enzyme prevent a beta-pyranose from binding in the same way. For the reverse reaction with ketoses, the anomeric specificity is less certain. Dideoxyimino-D-glucitol is a structural analogue of the ketose alpha-D-furanose. The binding of the inhibitor dideoxyimino-D-glucitol to the crystals of the enzyme does not mimic the binding of the reactive alpha-D-fructofuranose. Superposition of the nonphysiological substrate alpha-D-fructofuranose onto the atomic positions of dideoxyimino-D-glucitol is not possible due to the steric restrictions of the active site. However, by utilizing the approximate 2-fold symmetry of the sugar, a stereochemically sensible model is produced which is consistent with other data. In addition to reaction with alpha-D-furanose, the enzyme probably reacts with open ring keto sugars which are present at significant concentrations. Other sugars which resemble furanoses either do not inhibit significantly or are not observed in the crystals bound in a single conformation.     

Tight binding inhibitors of scytalone dehydratase: effects of site-directed mutations

We explore the use of site-directed mutations of scytalone dehydratase to study inhibitor binding interactions. The enzyme is the physiological target of new fungicides and the subject of inhibitor design and optimization. X-ray structures show that potent inhibitors (K(i)'s approximately 10(-)(11) M) interact mostly with 11 amino acid side chains and, in some cases, with a single backbone amide. Fifteen site-directed mutants of the 11 enzyme residues were prepared to disrupt enzyme-inhibitor interactions, and inhibition constants for 13 inhibitors were determined to assess changes in binding potencies. The results indicate that two of the six hydrogen bonds (always present in X-ray structures of native enzyme-inhibitor complexes) are not important for inhibitor binding. The other four hydrogen bonds are important for inhibitor binding, and the strength of the individual bonds is inhibitor-dependent. Inhibitor atoms remote from the hydrogen bonds influence their strength, presumably by effecting small changes in inhibitor orientation. Several hydrophobic amino acid residues are important recognition elements for lipophilic inhibitor functionalities, which is fully consistent with X-ray structures determined from crystals of enzyme-inhibitor complexes grown at neutral pH but not with those determined from crystals grown under acidic conditions. This study of mutant enzymes complements insights from X-ray structures and structure-activity relationships of the wild-type enzyme for refining views of inhibitor recognition.     

Fluoride inhibition of inorganic pyrophosphatase. I. Kinetic studies in a Mg2+-PPi system using a new continuous enzyme assay.

Reversible inhibition of bakers' yeast inorganic pyrophosphatase (EC 3.6.1.1) by fluoride has been studied as a function of substrate, metal-ion activator and inhibitor concentrations and pH using a new continuous enzyme assay with an automatic phosphate analyzer. The inhibition was shown to be the result of tight binding of fluoride by two catalytically active enzyme-substrate complexes. The reaction between pyrophosphatase and fluoride is relatively slow, so that the rate constants for the binding and release of the inhibitor were derived from phosphate formation curves measured on the time scale of enzyme assays. The pH-dependence of the inhibition reaction in the alkaline medium indicates that both the fluoride-enzyme interaction and the catalytic step of the pyrophosphatase reaction are controlled by the same group on the protein. In the acidic medium, the inhibition is considerably enhanced, presumably because of the protonation of another enzyme group.     

Synthesis and tyrosinase inhibitory activity of novel N-hydroxybenzyl-N-nitrosohydroxylamines

Several novel N-substituted N-nitrosohydroxylamines were synthesized. They all inhibited mushroom tyrosinase, but the type of inhibition was different depending on the substituent. Some N-(mono- or dihydroxybenzyl)-N-nitrosohydroxylamines exhibited uncompetitive inhibition with respect to L-dopa. Among them, compound 6 was also a competitive inhibitor with respect to oxygen. This observation suggests that another interaction by the meta- or para-hydroxyl group might stabilize the binding of the inhibitor to the enzyme through the oxygen binding site.     

Purification and specificity of a human microsomal epoxide hydratase

Epoxide hydratase was solubilized from human liver microsomal fractions and purified to an extent where the specific activity was 40-fold greater than that of the liver homogenate. Combination of homogenate and purified preparation showed that the increase in activity was not due to the removal of an inhibitor. Monosubstituted oxiranes with a lipophilic substituent larger than an ethyl group (isopropyl, t-butyl, n-hexyl, phenyl) readily interacted as substrates or inhibitors with this purified human epoxide hydratase, whereas those with a small substituent (methyl, ethyl, vinyl) were inactive, probably reflecting greater affinity of the former epoxides owing to lipophilic binding sites near the active site of the enzyme. In a series of oxiranes having a lipophilic substituent of sufficient size (styrene oxides), monosubstituted as well as 1,1- and cis-1,2-disubstituted oxiranes readily served as substrates or inhibitors of the enzyme, but not the trans-1,2-disubstituted, tri- or tetra-substituted oxiranes. trans-Substitution at the oxirane ring apparently prevents access of the oxirane ring to the active site by steric hindrance. Epoxide hydratase was also solubilized from microsomal fractions of rat and guinea-pig liver and purified by the same procedure. Structural requirements for effective interaction of substrates, inhibitors and activators were qualitatively identical for epoxide hydratase from the three sources. However, several quantitative differences were observed. Thus human hepatic epoxide hydratase seems to be very similar to, although not identical with, the enzyme from guinea pig or rat. Studies with epoxide hydratase from the latter two species therefore appear to be significant with respect to man. In addition, knowledge of structural requirements for epoxides to serve as substrates for human epoxide hydratase may prove useful for drug design. Compounds which need aromatic or olefinic moieties for their desired effect would not be expected to lead to accumulation of epoxides if their structure was such as to allow for a metabolically produced epoxide to be rapidly consumed by epoxide hydratase.     

Wild-type enzyme as a reporter of inhibitor binding by catalytically impaired mutant enzymes.

A method for the determination of inhibition constants for catalytically-debilitated mutant enzymes is described. The inhibitor is partitioned between the mutant and wild-type enzymes. Catalytic rates of the wild-type enzyme are used as the signal of inhibitor binding to the mutant enzyme. The method is validated with scytalone dehydratase, the Y50F mutant, and a potent inhibitor. The K(i) value for Y50F determined by this method is 0.49 +/- 0.10 nM. The K(i) value determined using the Y50F catalytic report for inhibitor binding in the absence of wild-type enzyme is 0.20 +/- 0.030 nM. The wild-type enzyme binds the inhibitor ten-fold less tightly, thus indicating that the hydrogen-bonding interaction between the Y50 hydroxyl group and the inhibitor (suggested by X-ray crystallography) is weak. The method is most useful when the catalytic activity of the wild-type enzyme is the most sensitive report of inhibitor binding and the mutant enzyme is greatly crippled in catalytic activity.     

Interaction of chicken cystatin with inactivated papains.

Papain which was inactivated by covalent attachment of small substituents to the active-site cysteine, up to the size of a carbamoylmethyl group, bound with high affinity to chicken cystatin (Kd less than approximately 15 pM), although less tightly than did active papain (Kd approximately 60 fM). However, as the size of the substituent was increased further, the affinity decreased appreciably, generally in proportion to the size of the inactivating group. For instance the dissociation constants for papain inactivated with N-ethylmaleimide and [N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-amido-(4-guanido )butane were 0.17 and approximately 10 microM respectively. The spectroscopic changes accompanying the reaction of all but the most weakly binding (Kd greater than or equal to 2 microM) inactivated papains with cystatin were similar to those induced by the active enzyme. Interactions involving the reactive cysteine residue of papain are thus not crucial for high-affinity binding of the enzyme to cystatin, in accordance with a recently proposed model for the enzyme-inhibitor complex, based on computer docking experiments. In this model there is sufficient space around the reactive cysteine in the complex for a small inactivating group, explaining the tight binding of papains with such substituents. However, larger inactivating groups cannot be accommodated in this space and therefore must displace the inhibitor out of the tight fit with the enzyme, in agreement with the observed decrease in binding affinity with increasing size of bulkier substituents. The kinetics of binding of cystatin to inactivated papains were compatible with simple, reversible, bimolecular reactions, having association rate constants of (7-9) x 10(6) M-1 s-1 at pH 7.4, 25 degrees C, similar to what was shown previously for the binding of cystatin to active papain. The rate of association of the inhibitor with either active or inactivated papain thus appears to be primarily diffusion-controlled. The decreasing affinity of cystatin for papains inactivated with groups of increasing size was shown to be due to progressively higher dissociation rate constants, consistent with the greater impairment of fit between the binding regions of the two molecules.     

Human liver alanine aminopeptidase. Inhibition by amino acids.

Human liver alanine aminopeptidase is inhibited by L-amino acids having hydrophobic side chains such as Phe, Tyr, Trp, Met, and Leu. Blocking of the amino group or the carboxyl group greatly reduces the inhibitory capacity of the amino acid. Kinetic studies demonstrate that inhibition of hydrolysis of the substrate L-Ala-beta-naphthylamide is of the noncompetitive type. Inhibition of the substrate L-Leu-L-Leu is of the mixed type. Inhibition of the substrate L-Ala-L-Ala-L-Ala is of the competitive type. These changes in the mechanism of inhibition are thought to be the result of the binding of the amino acid to the third residue binding site on the enzyme. This is the part of the active center to which the third residue from the amino end of a peptide substrate is normally bound. The inhibitor constants of several alanine oligopeptides are shown to decrease with increasing length through L-Ala-L-Ala-L-Ala-L-Ala, demonstrating that alanine aminopeptidase is a multisited enzyme with three and possibly four residue sites per active center. The inhibitor constant for Gly-Gly--Phe suggesting that indeed the third residue site preferentially binds large hydrophobic residues.     

NAD(P)+ analogues: tools for the investigation of the active site of oestradiol 17beta-dehydrogenase from human placenta.

Oestradiol-17beta:NAD+ 17-oxidoreductase from human placenta can accept coenzyme analogues of NAD+ and NADP+ where the amide group is replaced by methyl ketone, nitrile or thioamide. The inhibition with analogues of NAD+ has been studied. The presence of a substituent at C-3 of the pyridinium ring is necessary for the binding. The inhibition by C-4 methylated analogues is very poor, and the effect of a methyl group at C-5 depends on the substituent at C-3. The 1,4,5,6-tetrahydronicotinamide adenine dinucleotide is a competitive inhibitor. Nicotinamide 8-bromoadenine dinucleotide and nicotinamide 8-thioadenine dinucleotide are efficient hydrogen acceptors.     

Substrate form of D-frutose 1,6-bisphosphate utilized by fructose 1,6-bisphosphatase.

Rapid quench kinetic experiments on fructose 1,6-bisphosphatase demonstrate a stereospecificity for the alpha anomer of fructose 1,6-bisphosphate relative to the beta configuration. The beta anomer is only utilized after mutarotation to the alpha form in a process that is not enzyme catalyzed. Studies employing analogues of the acyclic keto configuration indicate that the keto form is utilized at a rate less than 5% that of the alpha anomer, a finding also confirmed by computer simulation of the rapid quench data. Chemical trapping experiments of the keto analogue, xylulose 1,5-bisphosphate, and the normal substrate suggest that interconversion of the acyclic and anomeric configurations is retarded by their binding to the enzyme. A hypothesis is advanced attributing substrate inhibition of fructose 1,6-bisphosphatase to possible binding of the keto species.     

Antiviral thiosemicarbazone and related compounds. III. Quantitative relationships between structure and antiviral activity of isantin-beta-isothio-semicarbazone against mengo virus]

The virostatic activity of isatin-beta-isothiosemicarbazones can be described quantitatively by hydrophobic, electronic, and steric substituent constants using multivariate regression analysis. A preceding Free-Wilson analysis allows data smoothening, and thus improved adaptation. Predictions made on the basis of the quantitative structure-action relationships obtained could be confirmed experimentally by synthesizing and testing the corresponding compounds.     

Structure-reactivity relationships for the inhibition mechanism at the second alkyl-chain-binding site of cholesterol esterase and lipase.

Alkyl-N-phenyl carbamates (2-8) (see Figure 1), alkyl-N-phenyl thiocarbamates (9-15), 2,2'-biphenyl-2-ol-2'-N-substituted carbamates (16-23), and 2, 2'-biphenyl-2-N-octadecylcarbamate-2'-N-substituted carbamates (24-31) are prepared and evaluated for their inhibition effects on porcine pancreatic cholesterol esterase and Pseudomona species lipase. All inhibitors are characterized as transient or pseudo substrate inhibitors for both enzymes. Both enzymes are not protected from inhibition and further inactivated by carbamates 2-8 and thiocarbamates 9-15 in the presence of trifluoroacetophenone. Therefore, carbamates 2-8 and thiocarbamates 9-15 are exceptions for active site binding inhibitors and are probably the second alkyl-chain binding-site-directed inhibitors for both enzymes. The inhibition data for carbamates 2-8 and thiocarbamates 9-15 are correlated with the steric constant, E(s), and the hydrophobicity constant, pi; however, the inhibition data are not correlated with the Taft substituent constant, sigma. A comparison of the inhibition data for carbamates 2-8 and thiocarbamates 9-15 toward both enzymes indicates that thiocarbamates 9-15 are more potent inhibitors than carbamates 2-8. A comparison of the inhibition data for cholesterol esterase and Pseudomona species lipase by carbamates 2-8 or thiocarbamates 9-15 indicates that cholesterol esterase is more sensitive to the E(s) and pi values than Pseudomona species lipase. The negative slope values for the logarithms of inhibition data for Pseudomona species lipase by carbamates 2-8 and thiocarbamates 9-15 versus E(s) and pi indicate that the second alkyl-chain-binding site of Pseudomona species lipase is huge, hydrophilic, compared to that of cholesterol esterase, and prefers to interact with a bulky, hydrophilic inhibitor rather than a small, hydrophobic one. On the contrary, the second alkyl-chain-binding site of cholesterol esterase prefers to bind to a small, hydrophobic inhibitor. Both enzymes are protected from inhibition by carbamates 16-23 in the presence of trifluoroacetophenone. Therefore, carbamates 16-23 are characterized as the alkyl chain binding site, esteratic site oxyanion active site directed pseudo substrate inhibitors for both enzymes. Both enzyme inhibition data for carbamates 16-22 are well-correlated with sigma alone. The negative rho values for these correlations indicate that the serine residue of both enzymes and carbamates 16-22 forms the tetrahedral species with more positive charges than inhibitors and the enzymes and follow the formation of the carbamyl enzymes with more positive charges than the tetrahedral species. Carbamates 24-31 are also exceptions for active site binding inhibitors and probably the second alkyl chain binding site-directed inhibitors for both enzymes. However, the enzyme inhibition constants for carbamates 24-31 are correlated with values of sigma, E(s), and pi. The negative rho values for these correlations indicate that both enzymes and carbamates 24-31 form the tetrahedral species with more positive charges than inhibitors and the enzymes and follow the formation of the carbamyl enzymes with more positive charges than those tetrahedral species. Therefore, carbamates 24-31 may bind to both the active sites and the second alkyl chain binding site and follow the evacuation of the active sites. A comparison of the rho values for cholesterol esterase and Pseudomona species lipase by carbamates 24-31 indicates that cholesterol esterase is much more sensitive to the sigma values than Pseudomona species lipase. The negative sensitivity values, delta, for the cholesterol esterase inhibitions by carbamates 24-31 indicate that the enzyme prefers to bind to a bulky carbamyl group rather than bind to a small one. The hydrophobicity of carbamates 24-31 does not play a major role in both enzyme inhibitions.     

Binding specificity of the two major DNA-binding proteins in human serum.

The two major DNA-binding proteins of human serum (DNA-binding protein 1 and DNA-binding protein 2) were shown to bind preferentially to single-stranded polynucleotides rich in guanine residues. Equilibrium competition experiments using a nitrocellulose filter assay system containing labeled human lymphocyte DNA and various competing natural and synthetic polynucleotides indicated that both proteins recognized sequences of bases containing a keto group in either position 6 (purines) or 4 (pyrimidines) and that these keto groups must be readily accessible for effective binding to occur. Guanine was shown to be the preferred nucleotide through inhibition experiments using a series of synthetic homopolymers and a series of bacterial DNAs of differing G + C content. The relationship between protein affinity and G + C content was shown to be directly proportional. The equilibrium constants for the binding of the human lymphocyte DNA by both proteins were on the order of 10(-6) M, and the length of the nucleotide sequence necessary for effective binding was found to be 12 to 18 bases using a series of oligomers of poly(dG).     

4-Amino derivatives of the Hsp90 inhibitor CCT018159

Novel piperazinyl, morpholino and piperidyl derivatives of the pyrazole-based Hsp90 inhibitor CCT018159 are described. Structure-activity relationships have been elucidated by X-ray co-crystal analysis of the new compounds bound to the N-terminal domain of human Hsp90. Key features of the binding mode are essentially identical to the recently reported potent analogue VER-49009. The most potent of the new compounds has a methylsulfonylbenzyl substituent appended to the piperazine nitrogen, possesses an IC50 of less than 600 nM binding against the enzyme and demonstrates low micromolar inhibition of tumour cell proliferation.     

Purification and properties of l(+)-lactate dehydrogenase from potato tubers

1. A purification of l(+)-lactate dehydrogenase is described. 2. The final preparation is active with NADH and NADPH and with a number of keto acids, but evidence is presented to support the view that a single enzyme is involved. 3. NAD(+) showed product inhibition, but at slightly acid pH values there was evidence of co-operative binding. 4. At acid pH values ATP was a potent inhibitor and appears to be an allosteric effector. At neutral or alkaline pH values ATP behaved as a weak competitive inhibitor. 5. The physiological significance of inhibition by ATP is discussed.     

Leaving group dependence in the phosphorylation of Escherichia coli alkaline phosphatase by monophosphate esters

Values of kcat and Km have been measured for the Escherichia coli alkaline phosphatase catalyzed hydrolysis of 18 aryl and 12 alkyl monophosphate esters at pH 8.00 and 25 degrees C. A Br?nsted plot of log (kcat/Km) (M-1 s-1) vs. the pK of the leaving hydroxyl group exhibits two regression lines: log (kcat/Km) = -0.19 (+/- 0.02) pKArOH + 8.14 (+/- 0.15) log (kcat/Km) = -0.19 (+/- 0.01) pKROH + 5.89 (+/- 0.17) Alkyl phosphates with aryl or large lipophilic side chains are not correlated by the above equations and occupy positions intermediate between the two lines. The observed change in effective charge on the leaving oxygen of the ester (-0.2) is very small, consistent with substantial electrophilic participation of the enzyme with this atom. Cyclohexylammonium ion is a noncompetitive inhibitor against 4-nitrophenyl phosphate substrate at pH 8.00, and neutral phenol is a competitive inhibitor (Ki = 82.6 mM); these data and the 100-fold larger reactivity of aryl over alkyl esters are consistent with the existence of a lipophilic binding site for the leaving group of the substrate. The absence of a major steric effect in kcat/Km for substituted aryl esters confirms that the leaving group in the enzyme--substrate complex points away from the surface of the enzyme. Arguments are advanced to exclude a dissociative mechanism (involving a metaphosphate ion) for the enzyme-catalyzed substitution at phosphorus.     

Functional interaction of diphenols with polyphenol oxidase. Molecular determinants of substrate/inhibitor specificity

Polyphenol oxidase (PPO) catalyzes the oxidation of o-diphenols to their respective quinones. The quinones autopolymerize to form dark pigments, an undesired effect. PPO is therefore the target for the development of antibrowning and antimelanization agents. A series of phenolic compounds experimentally evaluated for their binding affinity and inhibition constants were computationally docked to the active site of catechol oxidase. Docking studies suggested two distinct modes of binding, dividing the docked ligands into two groups. Remarkably, the first group corresponds to ligands determined to be substrates and the second group corresponds to reversible inhibitors. Analyses of the complexes provide structural explanations for correlating subtle changes in the position and nature of the substitutions on diphenols to their functional properties as substrates and inhibitors. Higher reaction rates and binding are reckoned by additional interactions of the substrates with key residues that line the hydrophobic cavity. The docking results suggest that inhibition of oxidation stems from an interaction between the aromatic carboxylic acid group and the apical His109 of the four coordinates of the trigonal pyramidal coordination polyhedron of CuA. The spatial orientation of the hydroxyl in relation to the carboxylic group either allows a perfect fit in the substrate cavity, leading to inhibition, or because of a steric clash flips the molecule vertically, facilitating oxidation. This is the first study to explain, at the molecular level, the determinants of substrate and inhibitor specificity of a catechol oxidase, thereby providing a platform for the design of selective inhibitors useful to both the food and pharmaceutical industries.     

Recognition of a cysteine substrate by E. coli gamma-glutamylcysteine synthetase probed by sulfoximine-based transition-state analogue inhibitors

A series of sulfoximine-based transition-state analogue inhibitors with a varying alkyl side chain was synthesized to probe the recognition of a Cys substrate by E. coli gamma-glutamylcysteine synthetase (gamma-GCS). The sulfoximines with a small alkyl group (H, methyl, ethyl, propyl, butyl and CH2OH) each served as a slow-binding inhibitor, the sulfoximine with an ethyl being by far the most potent inhibitor to cause facile and irreversible enzyme inhibition. As the size of the side chain changed from an ethyl, the inhibition potency markedly decreased to reduce the overall affinity with concomitant loss in the inactivation rate and with facile enzyme reactivation by dilution. The sulfoximine without a side chain inhibited the enzyme with almost the same potency as that of L-buthionine-(SR)-sulfoximine (BSO). The free energy difference calculated from the inhibition constants indicates that the side chain of Cys was recognized by its size through hydrophobic interaction and contributed almost equally or even more than the carboxy group to the overall binding of Cys in the transition state.