Studies Of Enzymatic Reactions Research Articles

Advances in capillary electrophoretically mediated microanalysis: An update

This review, as a continuation of an earlier report, gives an overview of recent developments, over the period from 2003 until now, in the use of capillary electrophoretic techniques for the in-line study of enzymatic reactions, derivatization, and chemical reactions. The article is divided into two parts: (i) in-line enzymatic reactions and (ii) in-line derivatization and chemical reactions. The first part introduces electrophoretically mediated microanalysis (EMMA) and discusses and illustrates the different modes of EMMA. A literature overview on enzymatic reactions is provided. The second part starts with an introduction of the procedures and the nomenclature used in the area of in-line derivatization and chemical reactions based on EMMA. Reported derivatization and chemical reaction procedures are discussed and summarized.

Synthesis of [2′‐2H1]‐Ribonucleosides

AbstractNew syntheses of C(2′)‐deuterated ribonucleosides have been accomplished starting either from 3,5‐di‐O‐benzyl‐1‐O‐methyl‐α,β‐D‐ribofuranose (1b) or 2,3‐O‐isopropylidene‐D‐ribose (14), with >97 atom‐% D incorporation in both cases. The former is suited to the demands of multiple‐site deuteration or uniform 13C/multiple 2H double labeling of the ribofuranose moiety, whereas the latter is particularly appropriate for single‐site 2H labeling for mechanistic studies of enzyme reactions.

Study of enzymatic reaction by electrophoretically mediated microanalysis in a partially filled capillary with indirect or direct detection.

Electrophoretically mediated microanalysis (EMMA), in combination with a partial filling technique and indirect or direct detection, is described for the study of enzymes reacting with the high mobility inorganic or organic anions as substrates or products. Part of the capillary is filled with a buffer optimized for the enzymatic reaction, the rest of the capillary with the background electrolyte being optimal for the separation of substrates and products. With haloalkane dehalogenase, chosen as a model enzyme, the enzymatic reaction was performed in a 20 mM glycine buffer (pH 8.6). Because of the wide substrate specificity of this enzyme, utilizing chlorinated as well as brominated substrates and producing either nonabsorbing chloride or absorbing bromide ions, two different background electrolytes and detection approaches were adopted. A 10 mM chromate-0.1 mM cetyltrimethylammonium bromide background electrolyte (pH 9.2) was used in combination with indirect detection and 20 mM beta-alanine-hydrochloric acid (pH 3.5) in combination with direct detection. The Michaelis constant (K(m)) of haloalkane dehalogenase for 1-bromobutane was determined. The K(m) values 0.59 mM estimated by means of indirect detection method and 0.17 mM by means of direct detection method were comparable with the value 0.13 mM estimated previously by gas chromatography.

Influence of Structural Fluctuation on Enzyme Reaction Energy Barriers in Combined Quantum Mechanical/Molecular Mechanical Studies

To account for protein dynamics and to investigate the effect of different conformations on the enzyme reaction energy barrier, we have studied the initial step of the acylation reaction catalyzed by acetylcholinesterase (AChE) with a multiple QM/MM reaction path approach. The approach consists of two main components: generating enzyme−substrate conformations with classical molecular dynamics simulation and mapping out the minimum reaction energy path for each conformational snapshot with combined quantum mechanical/molecular mechanical (QM/MM) calculations. It is found that enzyme−substrate conformation fluctuations lead to significant differences in the calculated reaction energy barrier; however, the qualitative picture of the role of the catalytic triad and oxyanion hole in AChE catalysis is very consistent. Our results emphasize the importance of employing multiple starting structures in the QM/MM study of enzyme reactions and indicate that structural fluctuation is an integral part of the enzyme reaction process.

Calculations of Activation Entropies of Chemical Reactions in Solution

The elucidation of the role of entropic effects in enzyme catalysis is a problem of practical and fundamental interest. In order to address this problem it is essential to develop simulation methods capable of evaluating the entropic contribution, (ΔS⧧)‘, of the reacting fragments to the total activation entropy, ΔS⧧. In fact, the general ability to evaluate activation entropies of chemical reactions in solution has long been a challenge to computational chemists. The present work develops and examines a method for evaluation of (ΔS⧧)‘ and ΔS⧧. This method introduces a thermodynamic cycle that considers the transformation between the reactants state (RS) and the transition state (TS) in two paths. In the first path the reacting fragments are constrained to move along a single reaction coordinate while in the second path they are allowed to move also in the subspace perpendicular to the reaction coordinate. The difference between the activation barriers that correspond to the two paths provides the desired −T(ΔS⧧)‘. The cycle also involves two steps where a Cartesian restraint that fixes the reacting fragments in the RS and TS is released. The free energies, ΔG‘'s, associated with these restraint release steps are used to complete the thermodynamic cycle and to provide the actual estimate of (ΔS⧧)‘. This estimate is optimized by using different initial conditions and by selecting the smallest value of |ΔG‘|. The solvent contributions to the activation entropy are evaluated using a newly developed version of the Langevin dipole model. The potential surfaces used in the present work are obtained by the empirical valence bond (EVB) method. This method provides analytical yet reliable potential surfaces that reflect properly the motions of the reacting fragments and the coupling between these motions and the solvent polarization. The analytical representation of the EVB surfaces allows us to perform the extensive sampling necessary in order to obtain converging results. Our method is examined by evaluating the activation entropy for the hydrolysis of formamide in water. It is found that the calculated activation entropies reach convergence in a reasonable computer time and that the agreement between the calculated and observed results is reasonable. This method can be easily implemented in studies of enzymatic reactions and helps in assessing the importance of entropic effects in enzyme catalysis.

Raman Study of Enzyme Reactions Using Potassium Ferricyanide as a Reaction Mediator: Quantitative Analysis of Substrates and Measurement of Enzyme Activity for Glucose Oxidase and Lactate Oxidase

This paper describes a Raman study of enzyme reactions that use potassium ferricyanide as a reaction mediator. The enzyme reaction systems investigated consisted of glucose oxidase (GOD), glucose, and potassium ferricyanide, and of lactate oxidase (LOD), lactate, and potassium ferricyanide. Raman spectra were measured for the above enzyme systems every 2 s to monitor the progress of the enzyme reactions at real time. The observed Raman spectra showed only three peaks at 2186, 2095, and 2022 cm−1 due to C N stretching modes of potassium ferricyanide (2186 cm−1) and potassium ferrocyanide (2095 and 2022 cm−1); with the progress of the enzyme reactions, the band at 2186 cm−1 decreases while those at 2095 and 2022 cm−1 increase. From the differentiation of the Raman intensity at 2022 cm−1, we could calculate the velocity ( V′) of the intensity change that directly reflects the rate ( V) of the enzyme reactions. By plotting the velocity thus obtained ( V′) vs. the concentration of the enzyme substrate ([ S]), we were able to develop a calibration curve to predict the concentration of the enzyme substrate. For GOD, the correlation coefficient ( R) and the detection limit of this calibration curve were 0.99 and 20 mg/dL, respectively. This detection limit was better than that obtained from conventional glucose sensors. For LOD, we were able to determine Michaelis constant ( Km) from the maximum velocity ( Vmax). The method proposed here is applicable to various kinds of enzymes that use potassium ferricyanide as a reaction mediator.

Nucleophilic substitution of halogen in 4-halo genated derivatives of glutamic acid

Previously, it was found that the reaction of dimethyl N-phthaloyl-4-bromoglutamate (1) with aniline derivatives proceeds diastereoselectively, affording the threoisomer as the major product. 2 Studying the factors determining the different reactivities of the diastereomers is of significant interest, because it might be useful for the synthesis of biologically active C(4)-derivatives of glutamic ac id) In addition, these compounds are used in mechanistic studies of enzymatic reactions. 4,5 In relation to these needs, we investigated the nucleophilic substitution of a halogen in 4-halogenated derivatives of glutamic acid. In the previous communication, 1 we proposed a mathematical model of the process and determined the rate constants for the reactions of the diastereomers of bromide 1 with p-anisidine in various solvents. It was shown that the reactions proceeded with the inversion of the configuration of C(4), and the mechanism of the substitution was close to the classical SN2. In the present work, the relative reactivities of diastereomers of bromide 1 and iodide 2 (a is threo, and b is erythro) in reactions with substituted anilines is studied (Scheme 1). Both the position of the substituent and its polarity were varied. The pseudo-first order rate constants k3 and k 4 and the parameter of diastereoselectively S = k4/k 3 of the reaction for diastereomers 1 and 2 were determined as previously described 1 in the presence of a 12-fold excess of the corresponding amine. The effect of polarity of para-substituents on the stereoselectivity was investigated in the following series:

Determination of enzyme activity by high-performance liquid chromatography.

The application of high-performance liquid chromatography (HPLC) in the study of enzymatic reactions is reviewed. The rationale for using HPLC is given and whether the components of the reaction mixture should be derivatized prior to or after HPLC. An alphabetical list of enzymes assayed by HPLC is given. Substrate and product are included as well the derivatization reagent, detection method and biological matrix. Specific examples of these assays in a complex biological matrix viz. faeces are given. Future prospects are the detection of new enzymes using synthetic substrates and implementation of mass spectrometry to elucidate enzyme specificities.

Computer simulations of enzymatic reactions: Current Opinion in Structural Biology 1992, 2:230–236

Computer-simulation approaches can shed light on the detailed relationship between the structure of proteins and their functions. The use of such approaches in studies of enzymatic reactions and related problems has increased significantly in recent years, and we are probably entering a period where the origin of the enormous catalytic power of enzymes will be gradually understood on a quantitative level. This review considers the main problems associated with realistic simulations of enzymatic reactions and the current state of this exciting field.

The Cyclopropyl Group in Studies of Enzyme Mechanism and Inhibition

AbstractThe cyclopropyl group—particularly in compounds such as cyclopropylmethanol, phenylcyclopropylamine, and cyclopropanone hydrate—has proved to be very useful in the study of enzyme reactions. Whether the cyclopropane ring is opened to form a radical or remains intact can provide much information about reactions occurring at the active site.

A secondary isotope effect in the lipoxygenase reaction

Fatty acids containing a prochiral tritium label have often been used in the study of enzymatic reactions which involve an obligatory step of hydrogen abstraction. In the lipoxygenase reaction, the primary isotope effect associated with this approach is detected as an isotopic enrichment of the substrate. Herein we characterize a previously unrecognized secondary isotope effect Which changes the specific activity of both the substrate and product. The 12-lipoxygenase of human platelets removes the 10- l s hydrogen of arachidonic acid in the formation of 12-hydroperoxyeicosatetraenoic acid. We studied the specific activity changes associated with conversion of the enantiomerically labeled [10- d R- 3H]arachidonic acid to 12-[10- 3H]hydroxyeicosatetraenoic acid in aspirin-treated platelets. [3- 14C]Arachidonic acid served as internal standard. The most pronounced change in 3H/ 14C ratio in the early stages of reaction was a 15–20% deficiency of tritium in the product. Later, the remaining arachidonate showed a marked increase in 3H/ 14C ratio. The changes in specific activity closely matched those predicted for a secondary isotope effect. Comparison of these data with the theoretical equations for a secondary isotope effect indicated the 10- d R- 3H substrate reacted at about 84% of the rate of unlabeled molecules. Interestingly, this secondary isotope effect is similar in magnitude to the secondary isotope effect in autoxidation reactions, a finding compatible with a basic similarity in reaction mechanisms in enzymatic and non-enzymatic oxygenation of lipids.

Studies of enzyme reactions in open systems

An experimental arrangement is described which allows studies of the temporal performance of enzyme reactions (also reactions of complexes of enzyme chains) under conditions of open systems. The various steady-state properties of such open systems can be recognized by measuring their input and output [1,2]. The various levels of flowing equilibria are perturbed by applying molecular modulators to the system, and the data obtained are compared to those found in closed systems. In order to prove the accuracy of the experimental configuration, the determination of K m in a one-substrate reaction is carried out in the open system, using only one concentration of substrate at various flow rates.

TEXTURAL CHANGES AND MOLECULAR CHARACTERISTICS OF PECTIC CONSTITUENTS IN RIPENING PEACHES

SUMMARY Pectic constituents were isolated from the alcohol insoluble solids of freestone peaches at four stages of ripeness. The varieties studied were Poppy, Loring, Southland and Rio Oso Gem. The pectic substances were fractionated into pectinic acids, pectic acids and protopectin fractions and the molecular weight of each fraction was calculated from its intrinsic viscosity value. The purity and degree of esterification of each fraction were determined. Corresponding shear press firmness values were also measured. Decrease in the molecular weight values of the pectinic acid fraction was found to be closely related to the increase in proportion of pectinic acids with advancing ripeness. A decrease in esterification of the pectic constituents with advancing ripeness was found mainly in the protopectin fraction. These changes were also closely related to the reduction in shear press firmness of the tissue during the ripening process in all varieties of freestone peaches studied. These results seem to provide a basis for studies of enzymatic reactions which influence fruit ripening.

Steps in the reactions of proteolytic enzymes with their substrates.

Kinetic experiments should be designed to answer specific questions about a reaction mechanism. The present paper is intended to show how a number of specific questions have been answered. Chymotrypsin and trypsin are mainly used to illustrate the different approaches, but many of the arguments used are equally applicable to the reactions of other hydrolytic enzymes with serine-OH or cysteine-SH at the active site. T he recognition of serine-OH and cysteine-SH as essential groups at the active sites of different hydrolytic enzymes did not rest on kinetic evidence. This was deduced from the correlation of enzyme activity with the extent of modification of specially reactive groups. The investigation of proton dissociation equilibria and the assignment of dissociation constants to groups with specified functions in substrate binding, catalysis or protein conformation was the first objective of serious kinetic studies of enzyme reactions. Steady state rate measurements over a wide range of pH showed that groups with p K 6.25 and 6.85 respectively are involved in the catalytic activity of trypsin and chymotrypsin with certain specific substrates (Hammond & Gutfreund 1955). In the case of chymotrypsin it was also shown by Hammond & Gutfreund (1955) that a group with a more alkaline pK is involved in substrate binding. This latter group was subsequently identified and its function was elucidated through the elegant experiments of Oppenheimer, Labouresse & Hess (1966). The identification of histidine as the group with p K A near neutrality, involved in the catalytic mechanism of trypsin and chymotrypsin, was subsequently confirmed by direct chemical methods by Schoelmann & Shaw (1963). Only kinetic analysis can demonstrate the involvement of proton donors or acceptors with specific properties in enzyme-substrate interaction or in catalysis. The clear identification of chemical groups capable of performing such functions is coming from the crystallographic analysis of the three-dimensional structure at the site of enzyme-substrate interaction, as illustrated in other papers presented in this discussion. Very interesting chemical information is obtained when the effect of structure on reactivity is synthesized from the composite of crystallographic and kinetic data.

Quantitative determination of methylindoles

A method is described for the quantitative determination of methylindoles, which have a free C 3, in the presence of tryptophan and methyltrytophans. The method, applicable also to indole, is particularly useful in the study of enzymic reactions.

I3. Catalysis in Biochemical Reactions

AbstractThe mechanism of enzyme action presents a formidable and challenging problem to the chemist and has spurred the development of rapid reaction methods towards the maximum in sensitivity and time resolution, in order to meet the stringent requirements of enzyme reactions. In this paper we will emphasize the ways in which rapid flow techniques have allowed the study of enzyme reactions in living cells and in isolated enzyme systems with various types of physical methods, some of which are inherently insensitive and sluggish.