Stereochemical aspects of peptide hydrolysis catalyzed by serine proteases of the chymotrypsin type

The steric course of peptide hydrolysis catalyzed by serine proteases has been studied on the basis of the available, extensive structural data and taking into account the stereoelectronic theory of Deslongchamps (Heterocycles, 7, 1271 (1977)). These studies allowed elucidation of the structure of intermediates, in particular of the tetrahedral intermediate, and of the main structural events taking place during catalysis. They reveal a difficulty inherent in the generally accepted mechanism of peptide hydrolysis: protonation of the leaving nitrogen in the configuration arising from nucleophilic attack of Ser-195 on the carbonyl carbon cannot take place internally from His-57. Two alternative mechanisms are discussed which are compatible with all implications of the stereoelectronic theory. The main features of the more probable mechanism are: (i) a conformational change allowing the imidazole ring of His-57 to occupy two distinct positions; in one position a proton is abstracted from O^γ of Ser-195, and in the other this proton is donated to the leaving nitrogen; (ii) a configurational change (inversion) of the pyramidal leaving nitrogen reorienting the lone-pair orbital developed during nucleophilic attack; in one orientation CO bond breaking, and in the other CN bond breaking, is allowed. This inversion process confers on the nitrogen the property of a switch controlling the breakdown of the tetrahedral intermediate.     

Kinetic alpha-deuterium isotope effects for enzymatic and nonenzymatic hydrolysis of nicotinamide-beta-riboside.

The kinetic α-secondary deuterium isotope effect, $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}$, for the pH-independent hydrolysis of nicotinamide riboside, yielding nicotinamide and ribose, in water at 25 ° is 1.14, establishing that this reaction proceeds with unimolecular substrate decomposition to yield a carboxonium ion, or related species, in the rate-determining step. Surprisingly, the corresponding isotope effect for the base-catalyzed decomposition of the same substrate is 1.12, a value indicating considerable sp² character at the Cl′ position in the transition state for this reaction. A similar result, $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}\text{= 1.15}$, was obtained for base-catalyzed hydrolysis of NAD⁺. The kinetic alpha deuterium isotope effect for the pig brain NAD glycohydrolasecatalyzed hydrolysis of nicotinamide riboside is 1.08. This value suggests that CN bond cleavage to form an intermediate carboxonium ion, or structurally related species, is at least partially rate-determining. In contrast, the corresponding value for the hydrolysis of this substrate catalyzed by Escherichia coli nicotinamide ribonucleotide glycohydrolase is very near unity, a result consistent with several interpretations including a rate-determining enzyme isomerization reaction.     

Proton nuclear magnetic resonance studies of histidine residues in rat and other rodent pancreatic ribonucleases. Effects of pH and inhibitors

Proton nuclear magnetic resonance spectra of the histidine residues in bovine and rat ribonuclease have been compared. The changes in chemical shift on titration and on binding of cytidine-3′-monophosphate and cytidine-2′-monophosphate have been followed. In the presence of the cytidine derivatives the spectra of both enzymes resemble each other more than those of the free enzymes. With these inhibitors, two histidines in rat ribonuclease exhibit the same pK values and shifts as the active site residues histidine 12 and 119 in the bovine enzyme. Their pK values in the inhibitor-free rat enzyme are about 0.4 higher than in the beef enzyme, which can be explained by the substitution at the entrance of the active site cleft of arginine 39 in the beef enzyme by serine in the rat enzyme. Rat ribonuclease contains one histidine with a rather high pK value of 7.6. The cytidine derivatives affect its chemical shift in exactly the same way as the shift of histidine 48 in bovine ribonuclease. The high pK value of this residue in rat ribonuclease can be explained by assuming a strong hydrogen bridge with glutamic acid 16. The other two histidines in rat ribonuclease have rather low pK values of 6.1 and 6.3. The histidine with a pK value of 6.3 has been assigned to position 105 and that with a pK value of 6.1 to position 73.The closer resemblance of the active sites of bovine and rat ribonuclease in the presence of inhibitors than in the inhibitor-free enzymes makes the concept of induced fit interesting from an evolutionary point of view.The characteristic downfield shift of the protonated form of histidine 119 in the complexes of bovine and rat ribonuclease with cytidine-3′-monophosphate is not observed with uridine-3′-monophosphate, suggesting non-identical binding of these pyrimidine nucleotides.Some preliminary results on the nuclear magnetic resonance properties of the histidine residues in coypu and chinchilla pancreatic ribonuclease have been obtained.     

Galactose-1-phosphate uridylyltransferase: identification of histidine-164 and histidine-166 as critical residues by site-directed mutagenesis

Galactose-1-phosphate uridylyltransferase catalyzes the interconversion of UDP-glucose and galactose-1-P with UDP-galactose and glucose-1-P by a double-displacement mechanism involving the compulsory formation of a uridylyl enzyme intermediate. The uridylyl group is covalently bonded to the N3 position of a histidine residue in the uridylyl enzyme. The galT gene of Escherichia coli, which codes for the uridylyltransferase and is contained in a plasmid for transformation of E. coli, has been sequenced, and the positions of the 15 histidine residues have been determined from the deduced amino acid sequence of this protein. Fifteen mutant genes, in each of which one of the 15 histidine codons has been changed to an asparagine codon, have been generated and used to transform the E. coli strain JM101. When extracts of the transformants were assayed for uridylyltransferase, 13 exhibited high levels of activity. Two of the extracts containing mutant uridylyltransferase exhibited less than control levels of activity. These mutant proteins, H164N and H166N, were overexpressed, isolated, and tested for their ability to form the compulsory uridylyl enzyme intermediate. Neither the H164N nor the H166N mutant proteins could form the intermediate. Thus, both His-164 and His-166 are critical for activity, and their proximity suggests that both are in the active site. One is the essential nucleophilic catalyst to which the uridylyl group is bonded in the intermediate, and the other serves an equally important, as yet unknown, function. The active-site sequence His(164)-Pro-His(166) is conserved in this enzyme from E. coli, humans, Saccharomyces, and Streptomyces.     

Catalysis of dehydrogenation of 4-trans-(N,N-dimethylamino)cinnamaldehyde by aldehyde dehydrogenase

4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) is a chromophoric and fluorogenic substrate of aldehyde dehydrogenase. Fluorescence of DACA is enhanced by binding to aldehyde dehydrogenase in the absence of catalysis both in the presence and absence of the coenzyme analogue 5′AMP. DACA binds to aldehyde dehydrogenase with a dissociation constant of 1–3 μM and stoichiometry of 2 mol mol⁻¹ enzyme. Incorporation of DACA during catalysis was also investigated and found to be 2 mol DACA mol⁻¹ enzyme. Effect of pH on the stoichiometry of DACA incorporation during catalysis has shown that DACA incorporation remained constant at 2 mol DACA mol⁻¹ enzyme, despite a 74-fold velocity enhancement between pH 5.0 and 9.0. Increase of pH increased decomposition of enzyme–acyl intermediate without affecting the rate-limiting step of the reaction. At pH 7.0 the pH stimulated velocity enhancement was 10-fold over that at pH 5.0; further velocity enhancement (11.5-fold that of pH 7.0) was achieved by 150 μM Mg²⁺ ions. The velocity at pH 7.0 with Mg²⁺ exceeded that of pH 9.0, and that at maximal pH stimulation at pH 9.5. It was observed that level of intermediate decreased to about 1 mol mol⁻¹ enzyme, indicating that Mg²⁺ ions increased the rate of decomposition of the enzyme–acyl intermediate and shifted the rate-limiting step of the reaction to another step in the reaction sequence.     

Genetic and biochemical characterization of Corynebacterium glutamicum ATP phosphoribosyltransferase and its three mutants resistant to feedback inhibition by histidine

ATP phosphoribosyltransferase (ATP-PRT) catalyzes the condensation of ATP and PRPP at the first step of histidine biosynthesis and is regulated by a feedback inhibition from product histidine. Here, we report the genetic and biochemical characterization of such an enzyme, HisG_Cg, from Corynebacterium glutamicum, including site-directed mutagenesis of the histidine-binding site for the first time. Gene disruption and complementation experiments showed that HisG_Cg is essential for histidine biosynthesis. HisG_Cg activity was noncompetitively inhibited by histidine and the α-amino group of histidine were found to play an important role for its binding to HisG_Cg. Homology-based modeling predicted that four residues (N215, L231, T235 and A270) in the C-terminal domain of HisG_Cg may affect the histidine inhibition. Mutating these residues in HisG_Cg did not cause significant change in the specific activities of the enzyme but resulted in the generation of mutant ones resistant to histidine inhibition. Our data identified that the mutant N215K/L231F/T235A resists to histidine inhibition the most with 37-fold increase in K_i value. As expected, overexpressing a hisG_Cg gene containing N215K/L231F/T235A mutations in vivo promoted histidine accumulation to a final concentration of 0.15 ± 0.01 mM. Our results demonstrated that the polarity change of electrostatic potential of mutant protein surface prevents histidine from binding to the C-terminal domain of HisG_Cg, resulting in the release of allosteric inhibition. Considering that these residues were highly conserved in ATP-PRTs from different genera of Gram-positive bacteria the mechanism by histidine inhibition as exhibited in Corynebacterium glutamicum probably represents a ubiquitously inhibitory mechanism of ATP-PRTs by histidine.     

Excess histidine enzymes cause AICAR-independent filamentation in Escherichia coli

High-level expression of the hisHAFI genes in Escherichia coli, cloned under the control of an IPTG-inducible promoter, caused filamentation, as previously reported in Salmonella typhimurium. We speculated that this filamentation might be produced by an action of the HisH and HisF enzymes on their product AICAR (amino-imidazole carboxamide riboside 5′-phosphate), a histidine by-product and normal purine precursor, possibly by favouring the formation of ZTP, the triphosphate derivative of AICAR. However, filamentation occured even in the absence of carbon flow through the histidine and purine pathways, as observed in a hisG purF strain lacking the first enzyme in each pathway. Filamentation thus does not require either the normal substrate or products of the overproduced histidine enzymes and must reflect another activity.     

Effects of nitrogen addition on litter decomposition, soil microbial biomass, and enzyme activities between leguminous and non-leguminous forests

Anthropogenic nitrogen (N) deposition is an expanding problem that affects the functioning and composition of forest ecosystems, particularly the decomposition of forest litters. Legumes play an important role in the nitrogen cycle of forest ecosystems. Two litter types were chosen from Zijin Mountain in China: Robinia pseudoacacia leaves from a leguminous forest (LF) and Liquidambar formosana leaves from a non-leguminous forest (NF). The litter samples were mixed into original forest soils and incubated in microcosms. Then, they were treated by five forms of N addition: NH₄ ⁺, NO₃ ^?, urea, glycine, and a mixture of all four. During a 6-month incubation period, litter mass losses, soil microbial biomass, soil pH, and enzyme activities were investigated. Results showed that mixed N and NO₃ ^?-N addition significantly accelerated the litter decomposition rates of LF leaves, while mixed N, glycine-N, and urea-N addition significantly accelerated the litter decomposition rates of NF leaves. Litter decomposition rates and soil enzyme activities under mixed N addition were higher than those under single form of N additions in the two forest types. Nitrogen addition had no significant effects on soil pH and soil microbial biomass. The results indicate that nitrogen addition may alter microbial allocation to extracellular enzyme production without affecting soil microbial biomass, and then affected litter decomposition process. The results further reveal that mixed N is a more important factor in controlling litter decomposition process than single form of N, and may seriously affect soil N cycle and the release of carbon stored belowground.     

Reaction of pyridoxal 5'-phosphate with Escherichia coli CoA transferase: evidence for an essential lysine residue.

The acetyl-CoA:acetoacetate CoA-transferase of Escherichia coli was reversibly inactivated by pyridoxal 5′-phosphate. The residual activity of the enzyme was dependent on the concentration of the modifying reagent to a concentration of 5 mm. The maximum level of inactivation was 89%. Kinetic and equilibrium analyses of inactivation were consistent with a two-step process (Chen and Engel, 1975, Biochem. J.149, 619) in which the extent of inactivation was limited by the ratio of first-order rate constants for the reversible formation of an inactive Schiff base of pyridoxal 5′-phosphate and the enzyme from a noncovalent, dissociable complex of the enzyme and modifier. The calculated minimum residual activity was in close agreement with the experimentally determined value. The conclusion that the loss of catalytic activity resulted from modification of a lysine residue at the active site was based on the following data, (a) After incubation with 5 mm pyridoxal 5′-phosphate, 3.95 mol of the reagent was incorporated per mole of free enzyme with 89% loss of activity, while 2.75 mol of pyridoxal 5′-phosphate was incorporated into the enzyme-CoA intermediate with a loss of 10% of catalytic activity; the intermediate was formed in the presence of acetoacetyl-CoA; (b) acid hydrolysis of the modified, reduced enzyme-CoA intermediate yielded a single fluorescent compound that was identified as N⁶-pyridoxyllysine by chromatography in two solvent systems; (c) the enzyme was also protected from inactivation by saturating concentrations of free CoA and ADP but not by adenosine. The results suggested that a lysine residue is involved in the electrostatic binding of the pyrophosphate group of CoA. Carboxylic acid substrate did not protect the enzyme from inactivation.     

Characterization of the four isomers of 123I-CMICE-013: A potential SPECT myocardial perfusion imaging agent

Myocardial perfusion imaging (MPI) with single photon emission computed tomography (SPECT) is widely used in the assessment of coronary artery disease (CAD). We have developed ¹²³I-CMICE-013 based on rotenone, a mitochondrial complex I (MC-1) inhibitor, as a promising new MPI agent. Our synthesis results in a mixture of four species of ¹²³I-CMICE-013 A, B, C, D. In this study, we separated the four species and evaluated their biodistribution and imaging properties. The cold analogs ¹²⁷I-CMICE-013 A, B, C, D were isolated and characterized and their chemical structures proposed. Methods: ¹²³I-CMICE-013 was synthesized by radiolabeling rotenone with Na¹²³I in trifluoroacetic acid (TFA) with iodogen as the oxidizing agent at 60 °C for 45 min, and the four species were separated by RP-HPLC. The cold analogs ¹²⁷I-CMICE-013 A, B, C and D were isolated with a similar procedure and characterized by NMR and mass spectrometry. Biodistribution and microSPECT imaging studies were carried out on normal rats. Results: We propose the mechanism of the rotenone iodination and the structures of the four species. First, I⁺ forms an intermediate three-membered ring with 6′ and 7′ carbons. Second, the lone electron pair of the water molecule attacks the 6′ or 7′-carbon, following by the formation of 6′-OH, and 7′-I bonds as in major products C and D, or 6′-I and 7′-OH bonds as in minor products A and B. The weaker 6′-I bond in the intermediate prompts the nucleophilic attachment of water at the favorable 6′-carbon to generate C and D. MicroSPECT images of ¹²³I-CMICE-013 A, B, C, D in rats showed clear visualization of myocardium and little interference from lung and liver. The imaging time activity curves and biodistribution data showed complex profiles for the four isomers, which is not expected from the structure activity relationship theory. Conclusion: ^123/127I-CMICE-013 A and B are constitutional isomers with C and D, while A and C are diastereomers of B and D, respectively. Overall, the biological characteristics of the four species are not correlated perfectly with their molecular structures.     

The reactivity of imidazole nitrogens in histidine to alkylation

Copper(II)-histidine complex was allowed to react at pH 6.0–6.1 at 22°C with bromoacetic acid. The reaction was followed by means of amino acid analysis of the histidine and N^im-carboxymethylhistidine derivatives. The results of the alkylation study indicate that the nucleophilic, active histidine molecule is coordinated to the copper(II) ion through the amino nitrogen and a carboxylate oxygen with the imidazole group turned away from the copper. This model of copper-bound histidine permitted the determination of the intrinsic nucleophilic activity of the imidazole nitrogens through their respective rate constants for alkylation. The tele-nitrogen is three times more reactive than the pros-nitrogen in the histidine and in the pros-carboxymethylhistidine-tele-carboxymethylhistidine systems. The carboxymethylation of copper(II)-histidine and bovine pancreatic ribonuclease have some analogies, which suggest that in pros-carboxymethylhistidine-119 ribonuclease the carboxylate unit of the alkylated histidine residue points into the active site.     

Purification and characterization of a mutant ATP phosphoribosyltransferase hypersensitive to histidine feedback inhibition.

A mutant form of ATP phosphoribosyltranferase (EC 2.4.2.17), hisG1708c, which results in abnormally slow growth of Salmonella typhimurium at 20 °C was purified to homogeneity and kinetic and chemical behavior were characterized. Initial velocity steady-state substrate kinetics of wild-type and mutant enzymes at 37 °C were consistent with sequential kinetics and demonstrated that standard assay concentrations of substrates were sufficient to substantially saturate both enzymes. Nearly time-independent inhibition by histidine at 37 °C could be obtained only after incubation in the presence of product and histidine. Studies at 37 °C showed that the mutant enzyme is 24 times more sensitive to histidine than the wild type in a negatively cooperative manner instead of the positively cooperative manner seen for wild type. Pure mutant enzyme exhibits two major electrophoretic species of native enzyme. Although one less cysteine is titratable in native mutant enzyme, the amino acid compositions of mutant and wild-type enzymes are similar. Histidine produces an ultraviolet difference spectrum in mutant enzyme closely resembling that produced in wild type. Binding of histidyl-tRNA to mutant enzyme is substantially inhibited by histidine. It is concluded that the hisG1708c mutation alters some conformational processes coupled to the histidine binding site while not affecting others.     

Mechanism of action of chalcone isomerase

The pH profile of the rate of isomerization of 4,2′,4′-trihydroxychalcone catalyzed by chalcone isomerase shows dependence on the basic form of a group with a pK of 7.25. The same pH dependence is seen for the reverse reaction. Enzyme activity is lost in the presence of diethylpyrocarbonate at pH 6.0. In the presence of 20% formamide in imidazole buffers, the pK for the forward reaction is modified by a second pK of 7.1. This behavior represents a perturbed pK of a neutral acid group and is attributable to the 2′ hydroxyl of the chalcone substrate. These results suggest a mechanism of enzyme action involving nucleophilic addition of an imidazole group in the active site to the double bond followed by nucleophilic attack by the 2′ phenolate group, resulting in ring closure with inversion of configuration at C-2.     

Specific features of distribution and size indices of four poorly studied species of sculpins (Cottidae) in the Okhotsk Sea Waters off Kamchatka

Evidence on occurrence in catches and characteristic of the spatial-bathymetric distribution and size indices of four species of Cottidae—frog sculpin Myoxocephalus stelleri, brightbelly sculpin Microcottus sellaris, antlered sculpin Enophrys diceraus, and furseal sculpin Stelgistrum stejnegeri—in summer-autumn months in the Okhotsk Sea waters off Kamchatka (site from 51°15′ to 57°20′ N, depths of 11–100 m) are provided. The first three species occur mainly in the northern part of the shelf above 54° N at depths smaller than 30–40 m within a comparatively well warmed surface water mass of seasonal modification at near-bottom temperature values above 6°C at various solid grounds. Maximum catches of S. stejnegeri were recorded only at a site of the western Kamchatka shelf from 54°00′ to 54°20′ N on pebbly-stony ground in a narrow bathy-metric range of 41–60 m on the boundary between the well warmed surface water mass of autumn modification and the cold intermediate water mass at a water temperature below 2°C. Evidence on the size-weight indices of the studied species of Cottidae in trawl catches in the Okhotsk Sea waters off Kamchatka in the study period is provided.     

Cultivation of a hybrid of free-living gametophytes between Undariopsis peterseniana and Undaria pinnatifida: morphological aspects and cultivation period

The kelp Undariopsis peterseniana is warm-water-tolerant, and consequently, there is currently considerable interest in developing commercial cultivation techniques for this species in Korea. U. peterseniana plants have been successfully transferred to the northern coast of Korea beyond their original habitat in Jeju Island (33°30′08.65″N, 126°55′39.02″E). In this study, we cultured a hybrid kelp consisting of a cross between free-living gametophytes of U. peterseniana and U. pinnatifida in an attempt to extend the culture period of Undaria which is an important species for both the abalone industry and for commercial seaweed mariculture for human food applications. Morphological characters and cultivation period were compared between the parent thalli and the hybrid. The cultivation experiment was conducted in Wando, on the southern coast of Korea (34°26′18.68″N, 127°05′43.88″E). The morphological characteristics of the hybrid thalli were intermediate between the two species having shallow pinnated blades and a reduced reproductive organ. Hybrid thalli showed faster growth rates, 1.5 times greater biomass, and a longer cultivation period than the parent thalli. The hybrid strain possessed characteristics that indicate it could be used as an alternative kelp source to supply the abalone feed industry.     

Direct Evidence for Nitrogen Ligation to the High Stability Semiquinone Intermediate in Escherichia coli Nitrate Reductase A

The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (Q_D) located in the vicinity of the distal heme b_D. Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. ¹⁴N and ¹⁵N ESEEM and HYSCORE measurements carried out at X-band (∼9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with ¹⁵N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant A_iso(¹⁴N) ∼0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using ¹⁴N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, ∼3.4 GHz), the ¹⁴N nuclear quadrupolar parameters of the interacting nitrogen nucleus (κ = 0.49, η = 0.50) were determined and correspond to those of a histidine N_δ, assigned to the heme b_D ligand His-66 residue. Moreover S-band ¹⁵N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T(¹⁵N) = 0.16 MHz). A distance of ∼2.2 Åbetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the Q_D site of NarGHI.     

The guanosine 5'-diphosphate D-mannose: guanosine 5'-diphosphate L-galactose epimerase of Chlorella pyrenoidosa. Chemical synthesis of guanosine 5'-diphosphate L-galactose and further studies of the enzyme and the reaction it catalyzes.

An enzyme from extracts of the green alga Chlorella pyrenoidosa that catalyzes the reversible epimerization of guanosine 5′-diphosphate d-mannose to guanosine 5′-diphosphate l-galactose was further purified. The substrate guanosine 5′-diphosphate l-galactose was made chemically by the morpholidate procedure. An improved method was developed for the synthesis of an intermediate in that process, β-l-galactopyranosyl phosphate, via an orthoester of l-galactose. Various characteristics of the enzyme and the reaction it catalyzes were studied. A new method using gas-liquid chromatography was introduced for following the course of the reaction with unlabeled substrates.     

Insights into the catalytic mechanism of human sEH phosphatase by site-directed mutagenesis and LC-MS/MS analysis

We have recently reported that human soluble epoxide hydrolase (sEH) is a bifunctional enzyme with a novel phosphatase enzymatic activity. Based on a structural relationship with other members of the haloacid dehalogenase superfamily, the sEH N-terminal phosphatase domain revealed four conserved sequence motifs, including the proposed catalytic nucleophile D9, and several other residues potentially implicated in substrate turnover and/or Mg²⁺ binding. To enlighten the catalytic mechanism of dephosphorylation, we constructed sEH phosphatase active-site mutants by site-directed mutagenesis. A total of 18 mutants were constructed and recombinantly expressed in Escherichia coli as soluble proteins, purified to homogeneity and subsequently analysed for their kinetic parameters. A replacement of residues D9, K160, D184 or N189 resulted in a complete loss of phosphatase activity, consistent with an essential function for catalysis. In contrast, a substitution of D11, T123, N124 and D185 leads to sEH mutant proteins with altered kinetic properties. We further provide evidence of the formation of an acylphosphate intermediate on D9 by liquid chromatography-tandem mass spectrometry based on the detection of homoserine after NaBH₄ reduction of the phosphorylated enzyme, which identifies D9 as the catalytic nucleophile. Surprisingly, we could only show such homoserine formation using the D11N mutant, which strongly suggests D11 to be involved in the acylphosphate hydrolysis. In the D11 mutant, the second catalytic step becomes rate limiting, which then allows trapping of the labile intermediate. Substrate turnover in the presence of ¹⁸H₂O revealed that the nucleophilic attack during the second reaction step occurs at the acylphosphate phosphorous. Based on these findings, we propose a two-step catalytic mechanism of dephosphorylation that involves the phosphate substrate hydrolysis by nucleophilic attack by the catalytic nucleophile D9 followed by hydrolysis of the acylphosphate enzyme intermediate supported by D11.     

Stereoselective chlorination of methyl β-lactoside with mesyl chloride in N,N-dimethylformamide

Treatment of methyl β-lactoside with mesyl chloride in N,N-dimethylformamide under a variety of conditions gave complex mixtures of chlorinated products, of which nine were isolated and characterised. Chlorination at a secondary position always occurred with inversion of configuration. When the reaction was conducted at 94° for 9 days, a mixture of the 3,3′,4′,6,6′-pentachloride, the 3,3′,6,6′- and 3,4′,6,6′-tetrachlorides, and the 3,6,6′- and 3′,6,6′-trichlorides was obtained together with the 3′,4′-epoxide of the 6,6′-dichloride, which was an artefact. Under milder conditions, the 6,6′-dichloride was encountered, together with methyl 6-chloro-6-deoxy-β-D-glucopyranoside which had arisen by hydrolysis of the interglycosidic bond. It is particularly noteworthy that displacement occurred at C-3′ of the lactoside, in spite of the vic-axial group at C-4′ which should hinder nucleophilic displacement at C-3′. The cause of this anomaly is discussed.     

The molecular mechanism of human hormone-sensitive lipase inhibition by substituted 3-phenyl-5-alkoxy-1,3,4-oxadiazol-2-ones

Hormone-sensitive lipase (HSL) plays an important role in the mobilization of free fatty acids (FFA) from adipocytes. The inhibition of HSL may offer a pharmacological approach to reduce FFA levels in plasma and diminish peripheral insulin resistance in type 2 diabetes. In this work, the inhibition of HSL by substituted 3-phenyl-5-alkoxy-1,3,4-oxadiazol-2-ones has been studied in vitro. 5-methoxy-3-(3-phenoxyphenyl)-1,3,4-oxadiazol-2(3H)-one (compound 7600) and 5-methoxy-3-(3-methyl-4-phenylacetamidophenyl)-1,3,4-oxadiazol-2(3H)-one (compound 9368) were selected as the most potent HSL inhibitors. HSL is inhibited after few minutes of incubation with compound 7600, at a molar excess of 20. This inhibition is reversed in the presence of an emulsion of lipid substrate. The reactivation phenomenon is hardly observed when incubating HSL with compound 9368. The molecular mechanism underlying the reversible inhibition of HSL by compound 7600 was investigated using high performance liquid chromatography and tandem mass spectrometry. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound per enzyme molecule. The inhibition by compound 7600 involves a nucleophilic attack by the hydroxy group of the catalytic Ser of the enzyme on the carbon atom of the carbonyl moiety of the oxadiazolone ring of the inhibitor, leading to the formation of covalent enzyme-inhibitor intermediate. This covalent intermediate is subsequently hydrolyzed, releasing an oxadiazolone decomposition product, carbon dioxide and the active HSL form. On the basis of this study, a kinetic model is proposed to describe the inhibition of HSL by compound 7600 in the aqueous phase as well as its partial reactivation at the lipid-water interface.