Richard L. Schowen

[29] Solvent isotope effects on enzyme systems

Publisher Summary This chapter discusses the solvent isotope effects on enzyme systems. The introduction of deuterium in place of protium in the hydrogenic sites of water, and its consequent exchange into some positions of enzymes and substrates produces solvent isotope effects on the kinetic and equilibrium constants associated with the enzymic reaction. These effects, usually expressed as ratios of the appropriate constants in the two isotopic solvents HOH and DOD, are useful in the study of reaction mechanism. The chapter provides (a) the physicochemical background for the use of solvent isotope effects in biochemical studies, with fairly complete derivations of the requisite algebraic expressions, an account of underlying assumptions, and a review of pertinent experimental and theoretical information. (b) to outline workable procedures for carrying out experiments in this area; and (c) to present the apparatus for interpretation of the results.

Transition States of Biochemical Processes

1 * Scope and Limitations of the Concept of the Transition State.- 2 * Catalytic Power and Transition-State Stabilization.- 3 * Quantum-Mechanical Approaches to the Study of Enzymic Transition States and Reaction Paths.- 4 * Primary Hydrogen Isotope Effects.- 5 * Secondary Hydrogen Isotope Effects.- 6 * Solvent Hydrogen Isotope Effects.- 7 * Heavy-Atom Isotope Effects in Enzyme-Catalyzed Reactions.- 8 * Magnetic-Resonance Approaches to Transition-State Structure.- 9 * Mapping Reaction Pathways from Crystallographic Data.- 10 * Transition-State Properties in Acyl and Methyl Transfer.- 11 * Transition States for Hydrolysis of Acetals, Ketals, Glycosides, and Glycosylamines.- 12 * Decarboxylations of ?-Keto Acids and Related Compounds.- 13 * The Mechanism of Phosphoryl Transfer.- 14 * Intramolecular Reactions and the Relevance of Models.- 15 * Transition-State Affinity as a Basis for the Design of Enzyme Inhibitors.- 16 * Transition-State Theory and Reaction Mechanism in Drug Action and Drug Design.- Author Index.

The proton inventory technique.

The proton inventory technique uses the dependence of enzymic reaction rate on the atom fraction of deuterium present in mixtures of protium oxide and deuterium oxide to deduce for simple cases the number of exchangeable hydrogenic sites that produce isotope effects, and the magnitude of the isotope effect generated at each site. For more complex cases, other information, such as the participation of more than a single step in limiting the rate, may be obtained. The background of the method, the conduct of the experiments and the interpretation of the results are briefly reviewed. The method is then illustrated in its application to various enzyme systems by a series of case histories.

Hydrogen bonds and proton transfer in general-catalytic transition-state stabilization in enzyme catalysis.

The question of the nature of the proton bridge involved in general acid-base catalysis in both enzymic and non-enzymic systems is considered in the light of long-known but insufficiently appreciated work of Jencks and his coworkers and of more recent results from neutron-diffraction crystallography and NMR spectroscopic studies, as well as results from isotope-effect investigations. These lines of inquiry lead toward the view that the bridging proton, when between electronegative atoms, is in a stable potential at the transition state, not participating strongly in the reaction-coordinate motion. Furthermore they suggest that bond order is well-conserved at unity for bridging protons, and give rough estimates of the degree to which the proton will respond to structural changes in its bonding partners. Thus if a center involved in general-catalytic bridging becomes more basic, the proton is expected to move toward it while maintaining a unit total bond order. For a unit increase in the pK of a bridging partner, the other partner is expected to acquire about 0.06 units of negative charge. The implications are considered for charge distribution in enzymic transition states as the basicity of catalytic residues changes in the course of molecular evolution or during progress along a catalytic pathway.

A Functional Assay for Quantitation of the Apparent Affinities of Ligands of P-Glycoprotein in Caco-2 Cells

AbstractPurpose. To develop a facile functional assay for quantitative determination of the apparent affinities of compounds that interact with the taxol binding site of P-glcoprotein (P-gp) in Caco-2 cell monolayers. Methods. A transport inhibition approach was taken to determine the inhibitory effects of compounds on the active transport of [3H]-taxol, a known substrate of P-gp. The apparent affinities (KI values) of the compounds were quantitatively determined based on the inhibitory effects of the compounds on the active transport of [3H]-taxol. Intact Caco-2 cell monolayers were utilized for transport inhibition studies. Samples were analyzed by liquid scintillation counting. Results. [3H]-Taxol (0.04 μM) showed polarized transport with the basolateral (BL) to apical (AP) flux rate being about 10-20 times faster than the flux rate in the AP-to-BL direction. This difference in [3H]-taxol flux could be totally abolished by inclusion of (±)-verapamil (0.2 mM), a known inhibitor of P-gp, in the incubation medium. However, inclusion of probenecid (1.0 mM), a known inhibitor for the multidrug resistance associated protein (MRP), did not significantly affect the transport of [3H]-taxol under the same conditions. These results suggest that P-gp, not MRP, was involved in taxol transport. Quinidine, daunorubicin, verapamil, taxol, doxorubicin, vinblastine, etoposide, and celiprolol were examined as inhibitors of the BL-to-AP transport of [3H]-taxol with resulting KI values of 1.5 ± 0.8, 2.5 ± 1.0, 3.0 ± 0.3, 7.3 ± 0.7, 8.5 ± 2.8, 36.5 ± 1.5, 276 ± 69, and 313 ± 112 μM, respectively. With the exception of that of quinidine, these KI values were comparable with literature values. Conclusions. This assay allows a facile quantitation of the apparent affinities of compounds to the taxol-binding site in P-gp; however, this assay does not permit the differentiation of substrates and inhibitors. The potential of drug-drug interactions involving the taxol binding site of P-gp can be conveniently estimated using the protocol described in this paper.

Catalytic Power and Transition-State Stabilization

This book is about the use of the transition-state concept, and of ideas about transition-state structure and the stabilization of transition states in catalysis, in understanding the rate processes of biochemistry. Essentially all the reactions considered are enzyme-catalyzed reactions or reactions studied in order to illuminate enzyme catalysis. In view of our thesis in this volume—that the transition state and its structure and interactions are the central feature of catalysis it is interesting to note that in much of the current writing on enzyme catalysis, the transition state is mentioned rather more rarely than might have been anticipated and its stabilization is sometimes relegated to the position of a single item in a long list of potential contributions to catalytic power. This is true in the pioneering volumes of Bruice and Benkovic,(1) in the monographs of Jencks(2) and Bender,(3) and in the recent reviews of Jencks(4) and Bruice.(5) It is likewise true of many other valuable books and articles, of which well over 5000 are cited in the sources just mentioned. Obvious exceptions to this custom are, of course, the authors interested in transition-state analogs, such as Lienhardt(6) and Wolfenden,t(7) but the general tendency is clear. There is certainly no agreement that all we need to know in order to understand enzyme catalysis is the structure of the transition state and the manner in which it is stabilized.

The photo-Favorskii reaction of p-hydroxyphenacyl compounds is initiated by water-assisted, adiabatic extrusion of a triplet biradical.

The p-hydroxyphenacyl group 1 is an effective photoremovable protecting group, because it undergoes an unusual photo-Favorskii rearrangement concomitant with the fast release (<1 ns) of its substrates in aqueous solution. The reaction mechanism of the diethyl phosphate derivative 1a was studied by picosecond pump−probe spectroscopy, nanosecond laser flash photolysis, and step−scan FTIR techniques. The primary photoproduct is a triplet biradical, 33, with a lifetime of about 0.6 ns. The release of diethyl phosphate determines the lifetime of the triplet state T1(1a), τ(T1) = 60 ps in wholly aqueous solution. Formation of a new photoproduct, p-hydroxybenzyl alcohol (6), was observed at moderate water concentrations in acetonitrile. It is formed by CO elimination from the elusive spirodione intermediate (4), followed by hydration of the resulting p-quinone methide (5). Computational studies show that CO elimination from the spirodione is a very facile process.

The degradation of carboplatin in aqueous solutions containing chloride or other selected nucleophiles

Abstract The degradation kinetics of carboplatin in aqueous solution in the presence and absence of chloride ions were studied at elevated temperatures (43–70 °C) at pH 7.0 and at various pH values at 70 °C. Reaction rates were then extrapolated to temperatures of interest (infusion storage temperature, ‘in-use’ infusion temperature and body temperature) from Arrhenius plots. Additionally, the effects of pH and added chloride, thiosulphate, thiocyanate, azide and iodide on the rate of degradation of carboplatin were determined in an attempt to obtain a greater insight into the mechanism of nucleophilic substitution of carboplatin.

Degradation of dacarbazine in aqueous solution

The effects of initial concentration (0.05-5.0 mg ml-1, 2.5 x 10(-4)-0.025 M) (pH 1-13), buffer concentration (0.01-0.075 M), light, antioxidants and co-solvents on the degradation of dacarbazine in aqueous solution were investigated at 37 degrees C. Liquid chromatography was used to monitor the degradation of dacarbazine as well as the appearance of degradation products. The kinetics of hydrolysis of dacarbazine in the dark were pseudo first-order and independent of the initial concentration of the drug. The degradation of dacarbazine was accelerated by light and at low concentration proceeded by pseudo zero-order kinetics. The pH-rate profiles showed that both the photolytic and the hydrolytic reactions were dependent on the ionization state of the molecule. The main degradation product of both hydrolysis and photolysis was detected by liquid chromatography and confirmed by mass spectrometry to be 2-azahypoxanthine.

A mechanistic and kinetic study of the E-ring hydrolysis and lactonization of a novel phosphoryloxymethyl prodrug of camptothecin.

AbstractPurpose. This study was done to determine the E-ring hydrolysis and lactonization mechanism of a water-soluble 20-phosphoryloxymethyl (POM) prodrug of camptothecin (P-CPT). Specifically, the role of the phosphate group in facilitating E-ring hydrolysis was examined. Methods. Resolution between the lactone and carboxylate forms of P-CPT and camptothecin (CPT) was achieved with a RPHPLC assay using UV-visible detection. E-ring P-CPT hydrolysis and lactonization kinetics were followed using 20 mM acetate or phosphate buffer (μ = 0.15 NaCl) over the pH range of 4 to 8 at 25.0°C. A kinetic solvent isotope effect (KSIE) study was used to further probe the mechanism of E-ring hydrolysis. Results. The hydrolysis and lactonization reactions followed pseudo-first-order kinetics in the approach to equilibrium. The equilibrium ratio of the open and closed forms of P-CPT was dependent on pH, with the closed form dominant at low pH and the open form dominant at high pH. Buffer concentration changes had little to no effect on the rate of P-CPT E-ring hydrolysis. The KSIE study provided an overall isotope effect of 2.47 and a proton inventory KSIE consistent with an intramolecular general base catalysis. Conclusions. P-CPT has a pH-dependent equilibrium between the lactone and carboxylate forms similar but not identical to that of CPT. The results suggest a hydrolysis reaction mechanism that involves a single site hydrogen exchange facilitated intramolecularly by the dianionic phosphate moiety of P-CPT via either general base catalysis of the lactone ring attack by water or breakdown of the tetrahedral intermediate.