On the active site of β-hexosaminidase from latex of Ficus glabrata

N-Acetyl-β-d-hexosaminidase (EC 3.2.1.52), active against both p-nitrophenyl-N-acetyl-β-d-glucosaminide and p-nitrophenyl-N-acetyl-β-d-galactosaminide, is present in latex of Ficus glabrata. The final specific activity was increased 150-fold from crude extract after ammonium sulphate fractionation and affinity chromatography on Concanavalin A-Sepharose. The activity ratio β-N-acetylglucosaminidase-β-N-acetylgalactosaminidase remained constant. Substrate competition, competitive inhibition studies and Arrhenius plots confirm that, in the hexosamidase, only one kind of active site is responsible for both activities. Acetate and acetamide are more effective competitive inhibitors than iodoacetamide, N-acetylglucosamine and N-acetylgalactosamine more than glucosamine and galactosamine, and α-methylmannoside more than mannose, suggesting that the active site binds the N-acetyl moiety of the substrate and a hydrophobic interaction of the methyl group is involved. The difference between the strength of the inhibition by mannosamine with respect to glucosamine and galactosamine, that do not inhibit, seems to be due to the position at C-2 of the amino group in the pyranose ring.     

A refined model of the sugar binding site of yeast hexokinase B.

The sugar binding site of monomeric yeast hexokinase B complexed with the competitive inhibitor o-toluoylglucosamine has been examined in the model resulting from a crystallographic refinement at 2·1 Å resolution. Difference Fourier maps calculated assuming various sugar configurations demonstrate that the o-toluoylglucosamine binds in the chair equatorial conformation with its 1-hydroxyl axial (α-anomer). The absence of a chemically derived amino acid sequence has complicated our interpretations of sugar-enzyme interactions. Nevertheless, we conclude that the carboxyl group of Asp189 is hydrogen-bonded to both the 6- and 4-hydroxyl groups. The 4-hydroxyl group is hydrogen-bonded also to Asx188 and Asx215, while the 3-hydroxyl is interacting with both Asx245 and Asx 188, consistent with the enzyme's observed sugar specificity. The carboxyl group of Asp 189 is excluded from solvent in the presence of glucose and may be acting as a general base to enhance the nucleophilicity of the 6-hydroxyl group and thereby promote its attack on the γ-phosphate of ATP.Glucose is shown to bind to the enzyme in the same orientation and conformation as the sugar moiety of o-toluoylglucosamine, so that the 6-hydroxyl group and the carboxyl of Asp 189 are in identical positions in complexes with these two sugars. The fact that o-toluoylglucosamine is not a substrate must be explained by two observations. First, the binding of glucose results in one lobe rotating by 12 ° relative to the other lobe, thereby closing off the slit into which the sugar has bound (Bennett &; Steitz, unpublished results). Second, o-toluoylglucosamine does not produce this conformational change, because the bulky toluoyl group prevents the closing of this slit between the two lobes. We conclude, therefore, that the large glucose-induced conformational change must be essential for subsequent catalytic steps.It appears unlikely from this study that thiols play any direct role in catalysis or in substrate binding. One thiol group, however, lies 5·5 Å from the 3-hydroxyl and is hydrogen-bonded to three of the Asx groups that are binding the sugar. Chemical modification of this buried thiol would disrupt the glucose binding site, which could account for the observation (Otieno et al., 1977) that cyanylation of one of the enzyme's thiols abolishes enzymatic activity.A sulfate molecule is bound to the enzyme by two serine side-chains and its sulfur atom is 5·5 Å from the 6-hydroxyl group of glucose. If the γ-phosphate of ATP binds to this sulfate binding site, it would still be a little too far from the 6-hydroxyl for direct phosphoryl transfer.     

S-(4-Bromo-2,3-dioxobutyl)CoA: an affinity label for certain enzymes than bind acetyl-CoA.

S-(4-Bromo-2,3-dioxobutyl)-CoA, a potential affinity label for enzymes possessing a receptor site(s) for short-chain acyl-CoA, was synthesized by condensing CoA and 1,4-dibromo-2,3-butanedione in acidified methanol. The new reagent was tested as an active site-directed irreversible inhibitor with four enzymes that accept a short-chain acyl-CoA as substrate. With citrate synthase (pig heart) and acetyl-CoA hydrolase (beef kidney) irreversible inhibition was observed, and the rate of inactivation obeyed first-order kinetics. Benzoyl-CoA, a reversible competitive inhibitor versus acetyl-CoA with both citrate synthase and acetyl-CoA hydrolase, protected the active site of both enzymes against the irreversible inhibitor. The new reagent was an exceptionally potent irreversible inhibitor of acetoacetyl-CoA thiolase (beef liver). Relatively low concentrations of the reagent (≥1 μm) completely inhibited the thiolase in less than 2 min. Preincubation of thiolase with acetoacetyl-CoA protected the enzyme against inhibition by S-(4-bromo-2,3-dioxobutyl)-CoA. In contrast, irreversible inhibition of l-3-hydroxyacyl-CoA dehydrogenase (pig heart) was not observed. Instead, the new reagent appeared to be a weak alternate substrate for this dehydrogenase. In all cases, the new reagent exhibited tight reversible binding at the active site since the measured K_i's (and K_m) were in the range, 30 to 120 μm. It is anticipated that the new reagent will be suitable for investigating a number of acyl-CoA using enzymes by affinity labeling techniques.     

Location of lysyl residues at the allosteric site of fructose 1,6-bisphosphatase

Various flavin analogs were used as alternate substrates or competitive inhibitors to characterize the FMN binding sites of the NADH- and NADPH-specific FMN oxidoreductases from Beneckea harveyi. Several polyhydroxyl compounds were found to be poor competitive inhibitors for the FMN sites of these enzymes. The FMN binding sites of the two enzymes were found to be quite similar. The NADH:FMN oxidoreductase binds FMN exclusively through the isoalloxazine ring. The methyl groups at positions 7 and 8 contribute significantly to this binding. Utilizing lumichrome as a competitive inhibitor of the FMN binding site and AMP as a competitive inhibitor of the NADH binding site, we were able to determine that the NADH:FMN oxidoreductase forms an active ternary complex with NADH binding first in an ordered mechanism. The NADPH oxidoreductase also binds FMN primarily through the isoalloxazine ring. Unlike their participation in reaction with the NADH-specfic enzyme, the methyl groups at positions 7 and 8 are not involved in binding. There was no significant binding of the ribityl phosphate moiety with either enzyme. Both enzymes have lower K_m values for lumiflavin than FMN.     

Modification of the active site of isocitrate lyase from Pseudomonas indigofera

Kinetic analysis of inactivation of isocitrate lyase from Pseudomonas indigofera by 3-bromopyruvate established that enzyme binds this compound prior to alkylation and that substrate, D_s-isocitrate, competes for the same site on the enzyme. The rate of inactivation was increased by EDTA which is a promoter of catalysis in the presence of activated (reduced) enzyme and substrate. The combination of products, glyoxylate plus succinate, also protected against inactivation. Glyoxylate plus itaconate, phosphoenolpyruvate, or maleate also protected. However, each of the latter three compounds or glyoxylate or succinate alone provided little or no protection. Pyruvate, a competitive inhibitor with respect to glyoxylate in the condensation reaction, also failed to protect. However, two dicarboxylates, meso-tartrate and oxalate, that are also competitive inhibitors with respect to glyoxylate provide some protection against inactivation by BrP perhaps by bridging across cationic sites that facilitate glyoxylate and succinate binding. These and other results imply that alkylation by 3-bromopyruvate occurs at the succinate part of the active site. A mechanism which includes a catalytic role for the cysteine residue at the active site is presented and discussed.     

Reaction mechanism of aspartate transcarbamylase from mouse spleen

The reaction mechanism of aspartate transcarbamylase from mouse spleen has been determined, using steady-state kinetics, isotope-exchange experiments, inhibition studies with a transition-state analog, and product-inhibition studies. Intersecting reciprocal plots obtained when one substrate was varied against different concentrations of the second substrate indicate that the mechanism is sequential. The transition-state analog, N-(phosphonacetyl)-l-aspartate, was a powerful inhibitor of aspartate transcarbamylase, with an inhibition constant (K_i) of 2.6 × 10^?8m at 37 °C and pH 7.4 in 0.05 m Na HEPES buffer. PALA gave competitive inhibition with carbamyl phosphate and noncompetitive inhibition with l-aspartate, indicating that carbamyl phosphate must bind before aspartate for catalysis to occur. A ping-pong mechanism in which carbamyl phosphate binds first was excluded by isotope-exchange experiments, since [³²P]inorganic phosphate was not incorporated into carbamyl phosphate in the absence of aspartate. Product-inhibition studies showed that only inorganic phosphate and carbamyl phosphate gave a competitive pattern; all other combinations of substrate and product gave noncompetitive inhibition patterns when incubations were carried out at subsaturating concentrations of the second substrate. These inhibition patterns showed that carbamyl phosphate binds first, aspartate binds second, carbamyl aspartate dissociates first, and phosphate dissociates second.     

Mechanistic and structural insights into the in vitro inhibitory action of hypericin on glutathione reductase purified from baker's yeast

This work aims at studying the interaction between glutathione reductase (GR) and hypericin. The type of inhibition was determined by measuring changes in GR activity at increasing concentrations of hypericin as well as at varying concentrations of glutathione disulfide (GSSG) and nicotinamide adenine dinucleotide phosphate (NADPH), and the binding pose of hypericin was predicted by molecular docking. Accordingly, hypericin emerges as an effective inhibitor of GR. When the variable substrate is GSSG, the type of inhibition is competitive. When the variable substrate is NADPH, however, the type of inhibition appears to be linear mixed‐type competitive. Our computational analyses suggest that hypericin binds in the large intermonomer cavity of GR, and that it may interfere with the normal positioning/functioning of the redox‐active disulfide center at the enzyme's active site. Overall, besides its contributory role in promoting oxidative stress via the formation of reactive oxygen species in photodynamic therapy, hypericin can also weaken cancer cells through inhibiting GR.     

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.     

GlcNAc-Thiazoline conformations

The title compound, a powerful inhibitor of retaining N-acetylhexosaminidases, can move freely among three pyranose solution conformations of similar energy—two twist boats and the ⁴C₁ chair—as revealed by NMR, calculational, and crystallographic studies. It binds in the enzyme active site only in the pseudo-⁴C₁ conformation, however, in which it most closely resembles the hypothetical bound substrate transition state, a ⁴E sofa that is approximately trigonal bipyramidal at the anomeric carbon.     

Inhibition Effects in the Hydrolysis Reactions of Esters and Peptides Catalyzed by Carboxypeptidase A: An Example of Cooperative Binding Effects with a Monomeric Enzyme

N-benzoyl-L-phenylalanyl-L-phenylalanine is an excellent peptide substrate for carboxy-peptidase A; at 30 degrees C and pH 7.5, K(m) is 2.6 x 10(-5) M while k(cat) is 177 s(-1) (k(cat)/K(m) = 6.8 x 10(6) M(-1) s(-1)). Indole-3-acetic acid is a noncompetitive or mixed inhibitor towards the peptide and toward hippuryl-L-phenylalanine; plots of E/V vs [Inhibitor] are linear. N-Benzoyl-L-phenylalanine is a competitive inhibitor of peptide hydrolysis, and plots of E/V vs [Inhibitor] are again linear. One molecule of inhibitor binds per active site, and these inhibitors bind in different sites. At constant peptide substrate concentration and a series of constant concentrations of indole-3-acetic acid, plots of E/V vs the concentration of N-benzoyl-L-phenylalanine are linear and intersect behind the E/V axis and above the [Inhibitor] axis. This shows that both inhibitors can bind simultaneously and that binding of one facilitates the binding of the other (beta = 0.18). Employing the ester substrate hippuryl-DL,beta-phenyllactate, the same type of behavior is observed in the reverse sense; N-benzoyl-L-phenylalanine is a linear noncompetitive inhibitor and indole-3-acetic acid is a linear competitive inhibitor. Again the two inhibitor plot is linear and intersects above the [Inhibitor] axis (beta = 0.12). Previous X-ray crystallographic studies have indicated that indole-3-acetic acid binds in the hydrophobic pocket of the S'(1) site, while N-benzoyl-L-phenylalanine binds in the S(1)-S(2) site. The product complex for hydrolysis of N-benzoyl-L-phenylalanyl-L-phenylalanine (phenylalanine + N-benzoyl-L-phenylalanine) occupies both of these sites. However, the present work shows that the peptide substrate does not bind to the enzyme at pH 7.5 so as to be competitive with indole-3-acetic acid. The binding sites may be formed via conformational changes induced or stabilized by substrate and product binding. Copyright 2000 Academic Press.     

Structural and mechanistic studies on N-(2-carboxyethyl)arginine synthase

N²-(2-Carboxyethyl)arginine synthase (CEAS), an unusual thiamin diphosphate (ThDP)-dependent enzyme, catalyses the committed step in the biosynthesis of the β-lactamase inhibitor clavulanic acid in Streptomyces clavuligerus. Crystal structures of tetrameric CEAS-ThDP in complex with the substrate analogues 5-guanidinovaleric acid (GVA) and tartrate, and a structure reflecting a possible enol(ate)-ThDP reaction intermediate are described. The structures suggest overlapping binding sites for the substrates d-glyceraldehyde-3-phosphate (d-G3P) and l-arginine, and are consistent with the proposed CEAS mechanism in which d-G3P binds at the active site and reacts to form an α,β-unsaturated intermediate, which subsequently undergoes (1,4)-Michael addition with the α-amino group of l-arginine. Additional solution studies are presented which probe the amino acid substrate tolerance of CEAS, providing further insight into the l-arginine binding site. These findings may facilitate the engineering of CEAS towards the synthesis of alternative β-amino acid products.     

Kinetics of glycogen phosphorylase a from the muscle of the blue crab, Callinectes danae

The kinetics of purified glycogen phosphorylase a from the muscle of the blue crab (Callinectes danae) were studied in the direction of glycogen synthesis, and in the direction of glycogen degradation with P_i or arsenate as substrates. The effects of AMP, UDPG, G-6-P, glucose, and arsenate on the appropriate systems were studied. AMP is an activator of the enzyme. Inhibition by UDPG with respect to P_i changes from noncompetitive to competitive when AMP is added; it changes from noncompetitive to mixed with respect to glycogen when AMP is added. G-6-P is a competitive inhibitor of G-1-P and arsenate. Inhibition by glucose with respect to glycogen changes from noncompetitive to competitive when AMP is added in the direction of glycogen breakdown; it is noncompetitive with respect to P_i. Arsenate is a competitive inhibitor with respect to P_i. The K_m for AMP increases in the presence of UDPG, and decreases with increasing concentrations of P_i or glycogen. We propose a model in which the enzyme bears three interacting sites: an active site, an activator (AMP) site, and an inhibitor (glucose) site. The active site has three subsites: one for P_i, one for glycogen, and one for a glucose moiety which may be part of the substrates or inhibitors.     

Competitive inhibition of Neurospora crassa chitin synthetase activity by tunicamycin.

Chitin synthetase from Neurospora crassa was inhibited in vitro by tunicamycin. The drug was found to be kinetically a linear competitive inhibitor (K_i ～ 480 μm) with respect to the substrate, UDP-N-acetylglucosamine. Since tunicamycin and UDP-N-acetylglucosamine are structurally similar and there exists linear competitive inhibition, it is likely that tunicamycin inhibits enzyme activity by directly competing with the substrate for access to the enzyme.     

Identification and comparison of the organophosphate acid anhydrase activities of the clam,Rangia cuneata

1. Tissue extracts of the commonly found brackish water clam Rangia cuneata were found to degrade the potent neurotoxin diisopropylfluorophosphate (DFP) and surprisingly N, N′-diisopropylphosphorodiamidofluoridate (mipafox).2. Results indicate two groups of molecular weight-estimates for substrate specific enzymes within the digestive gland of R. cuneata. When DFP was a substrate, a protein in the range of 30,500–21,300 D was identified as OPA anhydrase. With mipafox as substrate, an OPA anhydrase ranging in weight from 105,000 to 138,300 D was identified.3. This data suggests at least two forms of active OPA anhydrase type proteins are active within R. cuneata. Suggestions as to the natural role of the OPA anhydrases and the implications in predicting environmental toxicity and in hazardous waste site clean up are discussed.     

Characterisation of the endospermal trypsin inhibitor of barley

A trypsin inhibitor was isolated from grains of two row barley (cv. Proctor). The purified protein was identical with the corresponding inhibitor of a six row barley (cv. Pirkka); both proteins showed, a Pi of 7.4. The N-terminal amino acid was phenylalanine and an arginine residue was involved in the active site. Effects of substrate concentration showed that the inhibition was noncompetitive with a K_i of about 0.9 × 10^?7M. An enzyme-inhibitor complex was demonstrated by disc electrophoresis.     

Azolylthioacetamides as a potent scaffold for the development of metallo-β-lactamase inhibitors

In an effort to develop new inhibitors of metallo-β-lactamases (MβLs), twenty-eight azolylthioacetamides were synthesized and assayed against MβLs. The obtained benzimidazolyl and benzioxazolyl substituted 1–19 specifically inhibited the enzyme ImiS, and 10 was found to be the most potent inhibitor of ImiS with an IC₅₀ value of 15?nM. The nitrobenzimidazolyl substituted 20–28 specifically inhibited NDM-1, with 27 being the most potent inhibitor with an IC₅₀ value of 170?nM. Further studies with 10, 11, and 27 revealed a mixed inhibition mode with competitive and uncompetitive inhibition constants in a similar range as the IC₅₀ values. These inhibitors resulted in a 2–4-fold decrease in imipenem MIC values using E. coli cells producing ImiS or NDM-1. While the source of uncompetitive (possibly allosteric) inhibition remains unclear, docking studies indicate that 10 and 11 may interact orthosterically with Zn2 in the active site of CphA, while 27 could bridge the two Zn(II) ions in the active site of NDM-1 via its nitro group.     

Crystal structure of Drosophila melanogaster tryptophan 2,3-dioxygenase reveals insights into substrate recognition and catalytic mechanism

Tryptophan 2,3-dioxygenase (TDO) catalyzes the oxidative cleavage of the indole ring of l-tryptophan to N-formylkynurenine in the kynurenine pathway, and is considered as a drug target for cancer immunotherapy. Here, we report the first crystal structure of a eukaryotic TDO from Drosophila melanogaster (DmTDO) in complex with heme at 2.7 Å resolution. DmTDO consists of an N-terminal segment, a large domain and a small domain, and assumes a tetrameric architecture. Compared with prokaryotic TDOs, DmTDO contains two major insertion sequences: one forms part of the heme-binding site and the other forms a large portion of the small domain. The small domain which is unique to eukaryotic TDOs, interacts with the active site of an adjacent monomer and plays a role in the catalysis. Molecular modeling and dynamics simulation of DmTDO-heme-Trp suggest that like prokaryotic TDOs, DmTDO adopts an induced-fit mechanism to bind l-Trp; in particular, two conserved but flexible loops undergo conformational changes, converting the active site from an open conformation to a closed conformation. The functional roles of the key residues involved in recognition and binding of the heme and the substrate are verified by mutagenesis and kinetic studies. In addition, a modeling study of DmTDO in complex with the competitive inhibitor LM10 provides useful information for further inhibitor design. These findings reveal insights into the substrate recognition and the catalysis of DmTDO and possibly other eukaryotic TDOs and shed lights on the development of effective anti-TDO inhibitors.     

Thiolactomycin inhibits d-aspartate oxidase: A novel approach to probing the active site environment

d-Aspartate oxidase (DDO) and d-amino acid oxidase (DAO) are flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the oxidative deamination of d-amino acids. While several functionally and structurally important amino acid residues have been identified in the DAO protein, little is known about the structure–function relationships of DDO. In the search for a potent DDO inhibitor as a novel tool for investigating its structure–function relationships, a large number of biologically active compounds of microbial origin were screened for their ability to inhibit the enzymatic activity of mouse DDO. We discovered several compounds that inhibited the activity of mouse DDO, and one of the compounds identified, thiolactomycin (TLM), was then characterized and evaluated as a novel DDO inhibitor. TLM reversibly inhibited the activity of mouse DDO with a mixed type of inhibition more efficiently than meso-tartrate and malonate, known competitive inhibitors of mammalian DDOs. The selectivity of TLM was investigated using various DDOs and DAOs, and it was found that TLM inhibits not only DDO, but also DAO. Further experiments with apoenzymes of DDO and DAO revealed that TLM is most likely to inhibit the activities of DDO and DAO by competition with both the substrate and the coenzyme, FAD. Structural models of mouse DDO/TLM complexes supported this finding. The binding mode of TLM to DDO was validated further by site-directed mutagenesis of an active site residue, Arg-237. Collectively, our findings show that TLM is a novel, active site-directed DDO inhibitor that will be useful for elucidating the molecular details of the active site environment of DDO.     

Synthetic inhibitors of glucocerebroside β-glucosidase

The glucocerebrosidase of human placenta was studied with various potential inhibitors. Several compounds that resemble the lipoidal product of enzyme action, ceramide, proved to be excellent inhibitors, acting by mixed modes (primarily noncompetitively). These were N-decyl-dl-erythro-3-phenyl-2-amino-l, 3-propanediol and several p-substituted derivatives. These compounds were also highly effective in rat spleen toward glucocerebroside and p-nitrophenyl β-glucoside as substrates. The compounds were inactive toward the analogous enzyme, galactocerebrosidase of rat brain, and were slightly stimulatory toward the rat brain enzyme which makes galactocerebroside. Longer and shorter N-alkyl groups proved to be less effective. Decanoic acid amides of phenylaminopropanediol and related compounds proved to be relatively inert, although some were stimulatory. Deoxycorticosterone β-glucoside was a moderately effective noncompetitive inhibitor and is apparently hydrolyzed by a different glucosidase. p-Nitrophenyl β-glucoside was also a moderately effective inhibitor, acting by mixed modes. p-Chloromercuribenzenesulfonate was a good inhibitor, presumably acting on a sensitive cysteine residue. It is concluded that cerebrosidase contains two sensitive sites, one catalytic and the other allosteric, each containing an important anionic group and able to bind glucosides and ceramide-like compounds.     

Phenylethanolamine is a specific substrate for type B monoamine oxidase

The characteristics of phenylethanolamine as both a competitive inhibitor and as a substrate for monoamine oxidase (MAO) were studied using rat brain and liver homogenates. Although phenylethanolamine, even at high concentrations (1 mM), produced minimal inhibition of MAO when serotonin (a substrate for type A MAO) was used as the substrate, it was a potent competitive inhibitor (K_i=11 μM) of the deamination of phenylethylamine (a substrate for type B MAO). When phenylethanolamine was used as a substrate, deprenyl, a selective inhibitor of type B MAO, was found to produce a single sigmoid inhibition curve at low concentrations of the inhibitor (pI₅₀=7.5). These results indicate that phenylethanolamine is a specific substrate for type B MAO. Identification of the products formed under the assay conditions show that phenylethanolamine is converted to both mandelic acid and phenylethylene glycol by liver homogenates but only to the latter, neutral metabolite by brain homogenates.