Substrate inhibition in an enzymatic ordered bireactant system: non-linear modelling of the kinetics of hypoxanthine-guanine-phosphoribosyltransferase

Hypoxanthine-guanine-phosphoribosyltransferase (HGPRT; EC 2.4.2.8) was determined as an enzyme following an ordered bireaction in the presence of substrate inhibition due to hypoxanthine. This kind of inhibition has not been postulated for HGPRT so far. The mechanism and the kinetic constants of the reaction of HGPRT from Saccharomyces cerevisiae were investigated by initial velocity studies involving a non-linear regression analysis. In addition, two kinds of experimental designs were compared: the variation of hypoxanthine concentrations over wide ranges at different of fixed levels of 5-phosphoribosyl-1-pyrophosphate, and the use of five appropriate sets of experimental conditions each characterized by different hypoxanthine and 5-phosphoribosyl-1-pyrophosphate concentrations. Both experimental designs were consistent with an ordered bi bi mechanism including a dead-end-complex between the enzyme and hypoxanthine.     

Acyclic nucleoside phosphonates with unnatural nucleobases,favipiravir and allopurinol,designed as potential inhibitors of the human and Plasmodium falciparum 6-oxopurine phosphoribosyltransferases

Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is a key enzyme of the purine salvage pathway and its activity is crucial for the survival of certain parasites e.g. Plasmodium falciparum (Pf). Acyclic nucleoside phosphonates (ANPs) containing either guanine or hypoxanthine as the purine base are inhibitors of this enzyme. In this part of a SAR study, these two naturally-occurring nucleobases attached to an acyclic moiety were replaced by allopurinol or favipiravir. Both allopurinol and favipiravir ANPs were prepared via Mitsunobu reaction. The alkylation of favipiravir was optimized to yield both N- and O- regioisomers but the N-regioisomers were unstable under deprotection conditions. Thus, only the ANPs containing the O-isomer of favipiravir and those containing allopurinol were evaluated as potential inhibitors of human HGPRT and PfHGXPRT. Two ANPs with allopurinol as the base have K_i values of 10?μM and 30?μM for PfHGXPRT but do not inhibit human HGPRT activity at concentrations of 100–150?μM.     

Kinetics of befunolol reductase from rabbit liver

The kinetic mechanism for the reduction of befunolol catalyzed by befunolol reductase from rabbit liver was investigated. From the initial velocity analysis, product inhibition and coenzyme binding studies, the reduction of befunolol was found to proceed through an ordered Bi Bi mechanism, in which beta-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) binds to the enzyme firstly and NADP+ leaves lastly. NADPH bound to the free enzyme at a molar ratio of 1:1. Furthermore, the result of dead-end inhibition by Cibacron blue F3GA, a nucleotide analogue which binds to many enzymes, was consistent with the ordered Bi Bi mechanism for the enzyme.     

Differential Distortion of Purine Substrates by Human and Plasmodium falciparum Hypoxanthine‐Guanine Phosphoribosyltransferase to Catalyse the Formation of Mononucleotides

Plasmodium falciparum (Pf) hypoxanthine‐guanine phosphoribosyltransferase (HGPRT) is a potential therapeutic target. Compared to structurally homologous human enzymes, it has expanded substrate specificity. In this study, 9‐deazapurines are used as in situ probes of the active sites of human and Pf HGPRTs. Through the use of these probes it is found that non‐covalent interactions stabilise the pre‐transition state of the HGPRT‐catalysed reaction. Vibrational spectra reveal that the bound substrates are extensively distorted, the carbonyl bond of nucleobase moiety is weakened and the substrate is destabilised along the reaction coordinate. Raman shifts of the human and Pf enzymes are used to quantify the differing degrees of hydrogen bonding in the homologues. A decreased Raman cross‐section in enzyme‐bound 9‐deazaguanine (9DAG) shows that the phenylalanine residue (Phe186 in human and Phe197 in Pf) of HGPRT stacks with the nucleobase. Differential loss of the Raman cross‐section suggests that the active site is more compact in human HGPRT as compared to the Pf enzyme, and is more so in the phosphoribosyl pyrophosphate (PRPP) complex 9DAG–PRPP–HGPRT than in 9‐deazahypoxanthine (9DAH)–PRPP–HGPRT.     

The Kinetics Behavior of the Reduction of Formaldehyde Catalyzed by Alcohol Dehydrogenase (ADH) and Partial Uncompetitive Substrate Inhibition by NADH

Alcohol dehydrogenase (ADH) catalyzes the final step in the biosynthesis of methanol from CO₂. Here, we report the steady-state kinetics for ADH, using a homogeneous enzyme preparation with formaldehyde as the substrate and nicotinamide adenine dinucleotide (NADH) as the cofactor. When changing NADH concentrations with the fixed concentrations of HCHO (more or less than NADH), kinetic studies revealed a particular zigzag phenomenon for the first time. Increasing formaldehyde concentration can weaken substrate inhibition and improve catalytic efficiency. The kinetic mechanism of ADH was analyzed using the secondary fitting method. The double reciprocal plots (1/v～1/[HCHO] and 1/[NADH]) strongly demonstrated that the substrate inhibition by NADH was uncompetitive versus formaldehyde and partial. In the direction of formaldehyde reduction, ADH has an ordered kinetic mechanism with formaldehyde adding to enzyme first and product methanol released last. The second reactant NADH can combine with the enzyme–methanol complex and then methanol dissociates from it at a slower rate than from enzyme–methanol. The reaction velocity depends on the relative rates of the alternative pathways. The addition of NADH also accelerates the releasing of methanol. As a result, substrate inhibition and activation occurred intermittently, and the zigzag double reciprocal plot (1/v～1/[NADH]) was obtained.     

Voltammetric and impedance studies of inosine-5'-monophosphate and hypoxanthine

The oxidation mechanism and adsorption of inosine 5'-monophosphate and hypoxanthine were investigated in solutions of different pH using voltammetric and impedance methods at glassy carbon electrodes. For both compounds, the pH dependence from differential pulse voltammetry showed that the same number of electrons and protons are involved in the rate-determining step of the electrochemical reaction. In the case of hypoxanthine, it was also possible to study the effect of different concentrations. At high concentrations of hypoxanthine, two oxidation peaks were observed, the first due to hypoxanthine oxidation with formation of oligomers and the second due to hypoxanthine oligomer oxidation, both compounds adsorbing strongly. Impedance measurements corroborated the voltammetric results and enabled the study of the adsorption of hypoxanthine on glassy carbon.     

Substrate‐Guided Front‐Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide N‐Acetylgalactosaminyltransferase 2

The retaining glycosyltransferase GalNAc‐T2 is a member of a large family of human polypeptide GalNAc‐transferases that is responsible for the post‐translational modification of many cell‐surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum‐mechanics/molecular‐mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic‐electronic level of detail. Our study provides a detailed structural rationale for an ordered bi–bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front‐face S_Ni‐type reaction in which the substrate N‐acetyl sugar substituent coordinates efficient glycosyl transfer.     

The use of S,S-dialkyl dithiocarbonates in Li ion battery electrolytes

S,S-dialkyl dithiocarbonates such as S,S-dimethyl dithiocarbonate and S,S-diethyl dithiocarbonate were tested as solvents for rechargeable lithium ion batteries. Graphite electrodes can be successfully cycled at high reversible capacities in an S,S-dimethyl dithiocarbonate solution containing 1 M LiAsF₆, while lithium ions cannot intercalate into graphite in S,S-diethyl dithiocarbonate solutions. Fourier transform infrared spectroscopy, cyclic voltammetry and chronopotentiometric methods were used in order to monitor the reaction mechanism. Received: 23 April 1997 / Accepted: 5 June 1997     

Determination of total dissolved phosphorus in water samples by axial inductively coupled plasma atomic emission spectrometry

A procedure was developed to determine total dissolved phosphorus in groundwater-like samples using axial ICP-AES. Severe and peculiar matrix effects (strongly positive at lower and leveling off at higher matrix concentrations) in the presence of Na and Ca were observed. To reduce these matrix effects, a double approach was utilized consisting of a so-called minimum matrix in combination with an internal standard (Ga in this case). The ‘minimum matrix’ (small amounts of K, Mg and Na) was only added to the standard solution(s) used for the calibration. The detection limit for the whole procedure was 12 μg/L using the P213 nm line. Residual matrix effects were less than 3% (P213 nm line). Received: 21 February 1997 / Revised: 5 June 1997 / Accepted: 6 June 1997     

Determination of total dissolved phosphorus in water samples by axial inductively coupled plasma atomic emission spectrometry

A procedure was developed to determine total dissolved phosphorus in groundwater-like samples using axial ICP-AES. Severe and peculiar matrix effects (strongly positive at lower and leveling off at higher matrix concentrations) in the presence of Na and Ca were observed. To reduce these matrix effects, a double approach was utilized consisting of a so-called minimum matrix in combination with an internal standard (Ga in this case). The ‘minimum matrix’ (small amounts of K, Mg and Na) was only added to the standard solution(s) used for the calibration. The detection limit for the whole procedure was 12 μg/L using the P213 nm line. Residual matrix effects were less than 3% (P213 nm line). Received: 21 February 1997 / Revised: 5 June 1997 / Accepted: 6 June 1997     

Liquid chromatographic isolation of coplanar PCB congeners on an activated carbon stationary phase

A rapid and uncomplicated method for the fractionation of PCBs leading to an isolation of the highly toxic non-ortho substituted PCBs is described. The liquid chromatographic separation was achieved on a stationary phase consisting of activated carbon and Celite 545. Using eluents with different polarity, isolation of the non-ortho substituted PCBs in a single fraction was achieved. The fractions were analysed by GC/MS. The method was tested by the determination of non-ortho substituted PCBs in technical mixtures (Aroclor 1254 and Aroclor 1242). The results were compared with those obtained by using an HPLC fractionation on a porous graphitic carbon column. Finally, the micro-column fractionation was used for the determination of non-ortho substituted PCBs in native soil samples. Received: 5 March 1997 / Revised: 4 June 1997 / Accepted: 6 June 1997     

Acidity and proton affinity of hypoxanthine in the gas phase versus in solution: intrinsic reactivity and biological implications

Hypoxanthine is a mutagenic purine base that most commonly arises from the oxidative deamination of adenine. Damaged bases such as hypoxanthine are associated with carcinogenesis and cell death. This inevitable damage is counteracted by glycosylase enzymes, which cleave damaged bases from DNA. Alkyladenine DNA glycosylase (AAG) is the enzyme responsible for excising hypoxanthine from DNA in humans. In an effort to understand the intrinsic properties of hypoxanthine, we examined the gas-phase acidity and proton affinity using quantum mechanical calculations and gas-phase mass spectrometric experimental methods. In this work, we establish that the most acidic site of hypoxanthine has a gas-phase acidity of 332 +/- 2 kcal mol-1, which is more acidic than hydrochloric acid. We also bracket a less acidic site of hypoxanthine at 368 +/- 3 kcal mol-1. We measure the proton affinity of the most basic site of hypoxanthine to be 222 +/- 3 kcal mol-1. DFT calculations of these values are consistent with the experimental data. We also use calculations to compare the acidic and basic properties of hypoxanthine with those of the normal bases adenine and guanine. We find that the N9-H of hypoxanthine is more acidic than that of adenine and guanine, pointing to a way that AAG could discriminate damaged bases from normal bases. We hypothesize that AAG may cleave certain damaged nucleobases as anions and that the active site may take advantage of a nonpolar environment to favor deprotonated hypoxanthine as a leaving group versus deprotonated adenine or guanine. We also show that an alternate mechanism involving preprotonation of hypoxanthine is energetically less attractive, because the proton affinity of hypoxanthine is less than that of adenine and guanine. Last, we compare the acidity in the gas phase versus that in solution and find that a nonpolar environment enhances the differences in acidity among hypoxanthine, adenine, and guanine.     

Copper interference on the spectrophotometric determination of iron and their simultaneous determination using bathophenantroline-disulfonic acid disodium salt

The interference of copper on the spectrophotometric determination of iron with bathophenantroline-disulfonic acid disodium salt was studied using an experimental design. Copper interferes even below pH 5 [1], forming a yellow complex with bathophenantroline which turned to green after 5 min. This complex showed a maximum at 425 nm with a molar absorptivity of 7.5 × 10³ L mol^–1 cm^–1. Microgram quantities of iron and copper were determined simultaneously in water standard samples using bathophenantroline-disulfonic acid disodium salt at pH 4.8 and measuring the absorbances at two wavelengths. The interference of iron on the copper determination was also estimated. The RSDs of the method for both Cu and Fe were below 1.7%. Recoveries for Cu and Fe were within the ranges 97.2% to 98% and 99.7 to 100.5%, respectively. The method was applied to the determination of copper and iron in the waste water from a water treatment plant. The results obtained by spectrophotometry were compared with those obtained by flame AAS. Received: 8 July 1996 / Revised: 2 June 1997 / Accepted: 6 June 1997     

十二烷基磺酸钠对κ-卡拉胶溶胶-凝胶转变的影响

研究了阴离子表面活性剂十二烷基磺酸钠(sodium dodecyl sulfonate, SDSN)对同荷聚阴离子多糖κ-卡拉胶(κ-CAR)溶胶-凝胶转变的影响. 通过不同浓度的SDSN加入前后对κ-CAR溶液的流变行为和热行为的比较发现, SDSN随浓度不同基本以两种方式影响κ-CAR的溶胶-凝胶转变, 低浓度时(c＜5 mmol•L－1) SDSN对κ-CAR的溶胶-凝胶转变影响很小, 仅有轻微的促进作用, 使凝胶的起始温度和转变温度略向高温方向移动; 而当c＞5 mmol•L－1时, SDSN显著影响κ-CAR的溶胶-凝胶转变, 使转变温度向低温方向移动, 凝胶形成变得困难. 同时发现, SDSN的浓度大于其临界胶束浓度(CMC)时, 伴随κ-CAR的凝胶过程形成类似SDSN低温结晶的有序结构. 进一步利用生物染料亚甲基兰(MB)为探针, 通过测定κ-CAR-MB体系的UV-Vis光谱, 探讨了阴离子表面活性剂SDSN与聚阴离子κ-CAR相互作用的机理可能为同种电荷之间的静电排斥作用、对K＋的竞争以及疏水作用.     

Kinetics of inhibition of peroxidase activity of myeloperoxidase by quercetin

The inhibition of myeloperoxidase (MPO), isolated from human neutrophils, by quercetin was investigated by following peroxidase activity of the enzyme using o-dianisidine as the substrate. The inhibition parameters (IC₅₀) were obtained by graphical analysis of the inhibition curves. A reaction mechanism, which involved the enzyme inhibition by quercetin and H₂O₂ in excess, was proposed. The rate and equilibrium constants for the proposed reaction path were calculated from experimental data. Kinetic analysis in noninhibiting H₂O₂ concentration range in the absence and the presence of quercetin revealed that the reaction mechanism underwent Michaelis–Menten kinetics. K and V values indicated that quercetin was a mixed inhibitor of MPO activity. The initial reaction rates were recalculated using the obtained results. Calculated curves fitted the experimental results within the range of experimental error. © 2008 Wiley Periodicals, Inc. Int J Chem Kinet 40: 384–394, 2008     

A comparative QM/MM simulation study of the reaction mechanisms of human and Plasmodium falciparum HG(X)PRTases

QM/MM hybrid potential free-energy simulations are performed to compare the reaction mechanisms of human hypoxanthine guanine phosphoribosyl transferase (HGPRTase) and the corresponding enyzme from Plasmodium falciparum (Pf), hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRTase). These enzymes share 44% of sequence identity but display very different affinities for xanthine. The calculations show that in both enzymes phosphoribosyl transfer proceeds via a dissociative mechanism from an anionic form of the substrate. Nevertheless, there are significant differences in the geometries of critical structures along the reaction paths which it may be possible to exploit for the design of specific inhibitors against the Pf enzyme.     

Distribution of charge in the process of coating Fe powder by electrolysis in a fluidized bed

Theoretical calculations of the amount of Ni that is applied electrolytically in the form of a coating on Fe powder particles in a fluidized bed are based upon the assumption of distribution of charge between the surface of the solid cathode and the powder particles present in the working volume of the electrolyte. The experimentally measured data show good agreement with theory. The efficiency of the coating process is rather low because of inhibition of the process by hydroxocomplexes formed in the course of electrolysis. The inhibition effect becomes more significant in the case of high suspension density, small powder particles, high current density, and prolonged time of electrolysis. Received: 5 May 1997 / Accepted: 30 June 1997     

Quantitation of hypoxanthine in plasma from patients with ischemic heart disease: adaption of a high-performance liquid chromatographic method.

A high-performance liquid chromatographic method is described for the separation and quantitation of several purine compounds, including hypoxanthine. The isocratic separation of a standard mixture of nine compounds is achieved within 20 min on a reversed-phase Nucleosil 100-5C18 column, with a mobile phase of KH2PO4 (300 mM, pH 4.0)-methanol-acetonitrile-tetrahydrofuran (97.9:1:1:0.1, v/v). Uric acid, guanine, hypoxanthine, uridine, xanthine, allopurinol, inosine, guanosine and 7-methylxanthine were almost completely baseline-separated, with detection limits in the range 0.5-1.2 pmol per injection. The influence of the concentrations of buffer and tetrahydrofuran on the quality of separation are described. The within-day and the day-to-day precision were satisfactory (e.g. coefficients of variation of less than 1.5 and ca. 6.0%, respectively, for peak heights). The recovery of [3H]hypoxanthine added to samples was 86 +/- 1%. Hypoxanthine was quantified in human plasma samples obtained at various times during coronary artery bypass grafting. The hypoxanthine levels measured immediately after release of the aortic cross-clamp were significantly higher than those determined under control conditions (18.8 +/- 7.0 and 3.4 +/- 1.0 microM, respectively).     

Development of an enzyme-linked immunosorbent assay for gentamicin in human blood serum

An enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of gentamicin in human blood serum was developed. Peculiarities of the adsorption on the microtitre plate surface of the gentamicin-protein conjugate were investigated. Different conditions of the competition stage of the analysis were studied and conditions for gentamicin monitoring in human blood serum in the clinical range were optimized. The matrix effect on the assay results, the specificity of the analytical system and the stability of the reagents were examined. The method permits gentamicin concentrations to be determined in human blood serum, diluted 1/1000, in the linear range from 1 to 30 ng/mL. The assay is characterized by high sensitivity (0.5 ng/mL), good reproducibility (CV < 12%) and good correlation with PFIA (r = 0.943). Received: 17 June 1997 / Revised: 30 September 1997 / Accepted: 2 October 1997     

Development of an enzyme-linked immunosorbent assay for gentamicin in human blood serum

An enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of gentamicin in human blood serum was developed. Peculiarities of the adsorption on the microtitre plate surface of the gentamicin-protein conjugate were investigated. Different conditions of the competition stage of the analysis were studied and conditions for gentamicin monitoring in human blood serum in the clinical range were optimized. The matrix effect on the assay results, the specificity of the analytical system and the stability of the reagents were examined. The method permits gentamicin concentrations to be determined in human blood serum, diluted 1/1000, in the linear range from 1 to 30 ng/mL. The assay is characterized by high sensitivity (0.5 ng/mL), good reproducibility (CV < 12%) and good correlation with PFIA (r = 0.943). Received: 17 June 1997 / Revised: 30 September 1997 / Accepted: 2 October 1997