Analysis of UDP-galactose 4'-epimerase mutations associated with the intermediate form of type III galactosaemia

Type III galactosaemia is a hereditary disease caused by reduced activity in the Leloir pathway enzyme, UDP-galactose 4'-epimerase (GALE). Traditionally, the condition has been divided into two forms-a mild, or peripheral, form and a severe, or generalized, form. Recently it has become apparent that there are disease states which are intermediate between these two extremes. Three mutations associated with this intermediate form (S81R, T150M and P293L) were analysed for their kinetic and structural properties in vitro and their effects on galactose-sensitivity of Saccharomyces cerevisiae cells that were deleted for the yeast GALE homologue Gal10p. All three mutations result in impairment of the kinetic parameters (principally the turnover number, k(cat)) compared with the wild-type enzyme. However, the degree of impairment was mild compared with that seen with the mutation (V94M) associated with the generalized form of epimerase deficiency galactosaemia. None of the three mutations tested affected the ability of the protein to dimerize in solution or its susceptibility to limited proteolysis in vitro. Finally, in the yeast model, each of the mutated patient alleles was able to complement the galactose-sensitivity of gal10 Delta cells as fully as was the wild-type human allele. Furthermore, there was no difference from control in metabolite profile following galactose exposure for any of these strains. Thus we conclude that the subtle biochemical and metabolic abnormalities detected in patients expressing these GALE alleles likely reflect, at least in part, the reduced enzymatic activity of the encoded GALE proteins.

Tandem enzyme-catalysed reduction/cis-dihydroxylation of 2,2,2-trifluoroacetophenone: chemoenzymatic routes to new enantiopure phenol and benzylic alcohol reagents

Factors that control the competition between toluene dioxgenase-catalysed arene cis-dihydroxylation and dehydrogenase-catalysed ketone reduction have been studied, using whole cells of Pseudomonas putida UV and three alkylaryl ketones. The triol metabolite, obtained from 2,2,2-trifluoroacetophenone, has been used in the synthesis of single enantiomer chiral phenols and benzylic alcohols. Potential applications of the methylether derivatives of the chiral phenols and benzylic alcohols, as resolving agents, have been found. (c) 2007 Society of Chemical Industry.

Selectivity studies of the benzene cis-dihydrodiol dehydrogenase enzyme from Pseudomonas putida ML2 with Vicinal diol substrates

The enantiopure (1S, 2S)-cis-dihydrodiol metabolites 2B-5B have been obtained in low yield from the corresponding monosubstituted halobenzene substrates 2A-5A, using a wild-type strain of Pseudomonas putida (ML2) containing benzene dioxygenase (BDO). Benzene cis-dihydrodiol dehydrogenase (BCD) from P. putida ML2 and naphthalene cis-dihydrodiol dehydrogenase (NCD) from P. putida 8859 were purified and used in a comparative study of the stereoselective biotransformation of cis-dihydrodiol enantiomers 2B-5B. The BCD and NCD enzymes were found to accept cis-dihydrodiol enantiomers of monosubstituted benzene cis-dihydrodiol substrates 2B-5B of opposite absolute configuration. The acyclic alkene 1,2-diols 10-17 were also found to be acceptable substrates for BCD.

Characterisation of antibodies to chloramphenicol, produced in different species by enzyme-linked immunosorbent assay and biosensor technologies

Six polyclonal antisera to chloramphenicol (CAP) were successfully raised in camels, donkeys and goats. As a comparison of sensitivity, IC50 values ranged from 0.3 ng mL(-1) to 5.5 ng mL(-1) by enzyme-linked immunosorbent assay (ELISA) and from 0.7 ng mL(-1) to 1.7 ng mL(-1) by biosensor assay. The introduction of bovine milk extract improved the sensitivity of four of the antisera by ELISA and two by biosensor assay; a reduction in sensitivity of the remaining antisera ranged by a factor of 1.1-2.6. Porcine kidney extract reduced the sensitivity of all the antisera by a factor ranging from 1.1 to 7 by ELISA and a factor of 1.5 to 4 by biosensor. A low cross-reactivity with thiamphenicol (TAP) and florfenicol (FF) was displayed by antiserurn G2 (1.2% and 18%, respectively) when a homologous ELISA assay format was employed. No cross-reactivity was displayed by any of the antisera when a homologous biosensor assay format was employed. Switching to a heterologous ELISA format prompted three of the antisera to display more significant cross-reactivity with TAP and FF (53% and 82%, respectively, using Dl). The heterologous biosensor assay also increased the cross-reactivity of D1 for TAP and FF (56% and 129%, respectively) and of one other antiserum (Gl) to a lesser degree. However, unlike the ELISA, the heterologous biosensor assay produced a substantial reduction in sensitivity (by a factor of 6 for D1). (C) 2007 Elsevier B.V. All rights reserved.

Enhanced cAMP generation and insulin-releasing potency of two novel Tyr(1)-modified enzyme-resistant forms of glucose-dependent insulinotropic polypeptide is associated with significant antihyperglycaemic activity in spontaneous obesity-diabetes

Glucose-dependent insulinotropic polypeptide (GIP) is an important incretin hormone, which potentiates glucose-induced insulin secretion. Antihyperglycaemic actions of GIP provide significant potential in Type 11 diabetes therapy. However, inactivation of GIP by the enzyme dipeptidyl peptidase IV (DPP IV) and its consequent short circulating half-life limit its therapeutic use. Therefore two novel Tyr(1)-Modified analogues of GIP, N-Fmoc-GIP (where Fmoc is 9-fluorenylmethoxycarbonyl) and N-palmitate-GIP, were synthesized and tested for metabolic stability and biological activity. Both GIP analogues were resistant to degradation by DPP IV and human plasma. In Chinese hamster lung (CHL) cells expressing the cloned human GIP receptor, both analogues exhibited a 2-fold increase in cAMP-generating potency compared with native GIP (EC50 values of 9.4, 10.0 and 18.2 nM respectively). Using clonal BRIN-BD11 cells, both analogues demonstrated strong insulinotropic activity compared with native GIP (P <0.01 to P <0.001). In obese diabetic (ob/ob) mice, administration of N-Fmoc-GIP or N-palmitate-GIP (25 nmol/kg) together with glucose (18 mmol/kg) significantly reduced the peak 15 min glucose excursion (1.4- and 1.5-fold respectively; P <0.05 to P <0.01) compared with glucose alone. The area under the curve (AUC) for glucose was significantly lower after administration of either analogue compared with glucose administered alone or in combination with native GIP (1.5-fold; P <0.05). This was associated with a significantly greater AUC for insulin (2.1-fold; P <0.001) for both analogues compared with native GIP. A similar pattern of in vivo responsiveness was evident in lean control mice. These data indicate that novel N-terminal Tyr(1) modification of GIP with an Fmoc or palmitate group confers resistance to degradation by DPP IV in plasma, which is reflected by increased in vitro potency and greater insulinotropic and antihyperglycaemic activities in an animal model of Type 11 diabetes mellitus.

Kinetic analysis of the cleavage of human protease-activated receptor-1/2/3 and 4 using quenched-fluorescent peptide substrates

Protease-activated receptors [PARs] are a family of G-protein-coupled seven-transmembrane domain receptors that are activated by proteolytic cleavage of their amino-terminal exodomain. To characterize the cleavage rate of human PAR-1 / 2 / 3 and 4 by trypsin and thrombin, four synthetic quenched-fluorescent peptide substrates have been synthesized. Each substrate consisted of a ten-residue peptide spanning the receptor activation cleavage site and using progress-curve kinetics, k(cat)/K-m values were determined.

Characterization of the cellular and metabolic effects of a novel enzyme-resistant antagonist of glucose-dependent insulinotropic polypeptide

A novel N-terminally substituted Pro(3) analogue of glucose-dependent insulinotropic polypeptide (GIP) was synthesized and tested for plasma stability and biological activity both in vitro and in vivo. Native GIP was rapidly degraded by human plasma with only 39 +/- 6% remaining intact after 8 h, whereas (Pro(3))GIP was completely stable even after 24 h. In CHL cells expressing the human GIP receptor, (Pro(3))GIP antagonized the cyclic adenosine monophosphate (cAMP) stimulatory ability of 10(-7)M native GIP, with an IC50 value of 2.6 muM. In the clonal pancreatic beta cell line BRIN-BD11, (Pro(3))GIP over the concentration range 10(-13) to 10(-8) M dose dependently inhibited GIP-stimulated (10(-7) M) insulin release (1.2- to 1.7-fold; P <0.05 to P <0.001). In obese diabetic (ob/ob) mice, intraperitoneal administration of (Pro(3))GIP (25 nmol/kg body wt) countered the ability of native GIP to stimulate plasma insulin (2.4-fold decrease; P <0.001) and lower the glycemic excursion (1.5-fold decrease; P <0.001) induced by a glucose load (18 mmol/kg body wt). Collectively these data demonstrate that (Pro(3))GIP is a novel and potent enzyme-resistant GIP receptor antagonist capable of blocking the ability of native GIP to increase cAMP, stimulate insulin secretion, and improve glucose homeostasis in a commonly employed animal model of type 2 diabetes. (C) 2002 Elsevier Science (USA).

Extraction of electrode kinetic parameters from microdisc voltammetric data measured under transport conditions intermediate between steady-state convergent and transient linear diffusion as typically applies to room temperature ionic liquids

The extraction of electrode kinetic parameters for electrochemical couples in room-temperature ionic liquids (RTILs) is currently an area of considerable interest. Electrochemists typically measure electrode kinetics in the limits of either transient planar or steady-state convergent diffusion for which the voltammetic response is well understood. In this paper we develop a general method allowing the extraction of this kinetic data in the region where the diffusion is intermediate between the planar and convergent limits, such as is often encountered in RTILs using microelectrode voltammetry. A general working surface is derived, allowing the inference of Butler-Volmer standard electrochemical rate constants for the peak-to-peak potential separation in a cyclic voltammogram as a function of voltage scan rate. The method is applied to the case of the ferrocene/ferrocenium couple in [C(2)mim][N(Tf)(2)] and [C(4)mim][N(Tf)(2)].

Oxidation of several p-phenylenediamines in room temperature ionic liquids: Estimation of transport and electrode kinetic parameters

The electrochemical oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) has been studied by cyclic voltammetry and potential step chronoamperometry at 303 K in five ionic liquids, namely [C(2)mim] [NTf2], [C(4)mim] [NTf2] [C(4)mpyrr] [NTf2] [C(4)mim] [BF4], and [C(4)mim] [PF6] (where [C(n)mim](+) = 1-alkyl-3-methylimidazolium, [C(4)mpyrr](+) = N-butyl-N-methylpyrrolidinium, [NTf2](-) = bis(trifluoromethylsulfonyl)imide, [BF4](-) = tetrafluoroborate, and [PF6](-) = hexafluorophosphate). Diffusion coefficients, D, of 4.87, 3.32, 2.05, 1.74, and 1.34 x 10(-11) m(2) s(-1) and heterogeneous electron-transfer rate constants, k(0), of 0.0109, 0.0103, 0.0079, 0.0066, and 0.0059 cm s(-1) were calculated for TMPD in [C(2)mim] [NTf2], [C(4)mim] [NTf2], [C(4)mpyrr] [NTf2], [C(4)mim] [BF4], and [C(4)mim] [PF6], respectively, at 303 K. The oxidation of TMPD in [C4mim][PF6] was also carried out at increasing temperatures from 303 to 343 K, with an activation energy for diffusion of 32.3 kJ mol(-1). k(0) was found to increase systematically with increasing temperature, and an activation energy of 31.4 kJ mol(-1) was calculated. The study was extended to six other p-phenylenediamines with alkyl/phenyl group substitutions. D and k(0) values were calculated for these compounds in [C(2)mim] [NTf2], and it was found that k(0) showed no obvious relationship with the hydrodynamic radius, r.

Kinetic analysis of the reaction between electrogenerated superoxide and carbon dioxide in the room temperature ionic liquids 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide and hexyltriethylammonium bis(trifluoromethylsulfonyl)imide

The reduction of oxygen in the presence of carbon dioxide has been investigated by cyclic voltammetry at a gold microdisk electrode in the two room-temperature ionic liquids 1-ethyl-3-methylimidazolium bis-(trifluoromethylsulfonyl)imide ([EMIM][N(Tf)(2)]) and hexyltriethylammonium bis(trifluoromethylsulfonyl)imide ([N-6222] [N(Tf)(2)]). With increasing levels of CO2, cyclic voltammetry shows an increase in the reductive wave and diminishing of the oxidative wave, indicating that the generated superoxide readily reacts with carbon dioxide. The kinetics of this reaction are investigated in both ionic liquids. The reaction was found to proceed via a DISP1 type mechanism in [EMIM][N(Tf)(2)] with an overall second-order rate constant of 1.4 +/- 0.4 x 10(3) M-1 s(-1). An ECE or DISP1 mechanism was determined to be the most likely pathway for the reaction in [N-6222][N(Tf)(2)], with an overall second-order rate constant of 1.72 +/- 0.45 x 10(3) m(-1) s(-1).

Trends in C-O and C-N bond formations over transition metal surfaces: An insight into kinetic sensitivity in catalytic reactions

Transition metal catalyzed bond formation is a fundamental process in catalysis and is of general interest throughout chemistry. To date, however, the knowledge of association reactions is rather limited, relative to what is known about dissociative processes. For example, surprisingly little is known about how the bond-forming ability of a metal, in general, varies across the Periodic Table. In particular, the effect of reactant valency on such trends is poorly understood. Herein, the authors examine these key issues by using density functional theory calculations to study CO and CN formations over the 4d metals. The calculations reveal that the chemistries differ in a fundamental way. In the case of CO formation, the reaction enthalpies span a much greater range than those of CN formation. Moreover, CO formation is found to be kinetically sensitive to the metal; here the reaction barriers (E-a) are found to be influenced by the reaction enthalpy. CN formation, conversely, is found to be relatively kinetically insensitive to the metal, and there is no correlation found between the reaction barriers and the reaction enthalpy. Analysis has shown that at the final adsorbed state, the interaction between N and the surface is relatively greater than that of O. Furthermore, in comparison with O, relatively less bonding between the surface and N is observed to be lost during transition state formation. These greater interactions between N and the surface, which can be related to the larger valency of N, are found to be responsible for the relatively smaller enthalpy range and limited variation in E-a for CN formation. (C) 2007 American Institute of Physics.

Enzyme-catalysed synthesis and reactions of benzene oxide/oxepine derivatives of methyl benzoates

A series of twelve benzoate esters was metabolised, by species of the Phellinus genus of wood-rotting fungi, to yield the corresponding benzyl alcohol derivatives and eight salicylates. The isolation of a stable oxepine metabolite, from methyl benzoate, allied to evidence of the migration and retention of a carbomethoxy group ( the NIH Shift), during enzyme-catalysed ortho-hydroxylation of alkyl benzoates to form salicylates, is consistent with a mechanism involving an initial arene epoxidation step. This mechanism was confirmed by the isolation of a remarkably stable, optically active, substituted benzene oxide metabolite of methyl 2-( trifluoromethyl) benzoate, which slowly converted into the racemic form. The arene oxide was found to undergo a cycloaddition reaction with 4-phenyl-1,2,4-triazoline-3,5-dione to yield a crystalline cycloadduct whose structure and racemic nature was established by X-ray crystallography. The metabolite was also found to undergo some novel benzene oxide reactions, including epoxidation to give an anti-diepoxide, base-catalysed hydrolysis to form a trans-dihydrodiol and acid-catalysed aromatisation to yield a salicylate derivative via the NIH Shift of a carbomethoxy group.