基于分子机制的蛋白质酶解反应经验修饰动力学模型

蛋白质酶解反应动力学行为的复杂性在于体系中底物与产物的多样性,以及由此决定的动力学常数的可变性.基于此,以酪蛋白(case in)—胰蛋白酶(trypsin)为模式体系,拟合求得动力学常数(Km和kcat)随水解度(DH)值变化的函数表达式,其规律为:随DH值增加,Km增大,kcat减小,kcat/Km减小,证明:酶与底物的亲和力随肽链缩短而减小,即高分子量多肽为蛋白酶的适宜底物,而酶解效率与酶解专一性随反应进行逐渐降低.进一步,根据分子水平的蛋白质酶解作用机制,关联水解实验数据,得到case in-trypsin酶解反应的经验修饰动力学方程(模型平均相对误差<5%),为定量表征复杂酶解历程以及高效制备活性多肽提供了理论基础.     

Phosphate triester hydrolysis promoted by an N2S(thiolate)Zn complex: mechanistic implications for the metal-dependent reactivity of peptide deformylase

The zinc(II) complex (PATH)ZnOH, where PATH is an N2S(thiolate) ligand, has been investigated for its ability to promote the hydrolysis of the phosphate triester tris(4-nitrophenyl) phosphate (TNP). The hydrolysis of TNP was examined as a function of PATH-zinc(II) complex concentration, substrate concentration, and pH in a water/ethanol mixture (66:33 v/v) at 25 degrees C. The reaction is first order in both zinc(II) complex and substrate, and the second-order rate constants were derived from linear plots of the observed pseudo-first-order rate constants versus zinc complex concentration at different pH values. A pH-rate profile yielded a kinetic pK(a) of 8.52(5) for the zinc-bound water molecule and a pH-independent rate constant of 16.1(7) M(-1) s(-1). Temperature-dependent studies showed linear Eyring behavior, yielding the activation parameters DeltaH++ = 36.9(1) kJ mol(-1) and DeltaS++ = -106.7(4) J mol(-1) K(-1). Interpretation of the kinetic data leads to the conclusion that hydrolysis of TNP takes place through a hybrid mechanism, in which the metal center plays a dual role of providing a nucleophilic hydroxide and activating the substrate through a Lewis acid effect. The synthesis and structural characterization of the related nickel(II) and iron(II) complexes [(PATH)2Ni2]Br2 (2) and (PATH)2Fe2Cl2 (3) are also described. Taken together, these data suggest a possible explanation for the low reactivity of the zinc(II) form of peptide deformylase as compared to the iron(II) form.     

Hydrolysis of N-Cyanoazoles

The hydrolysis kinetics of N-cyanoazoles in alkaline solutions is described by a first-order kinetic equation with respect to the substrate and concentration of hydroxide ions. The hydrolysis rate constants increase with increasing number of nitrogen atoms in the heterocyclic moiety and decrease with introduction of the annelated benzene ring.     

Kinetic studies of the urease-catalyzed hydrolysis of urea in a buffer-free system

The kinetics of urea hydrolysis catalyzed by urease, mainly in the absence of buffers by use of the self-buffer effect of the products, was investigated. The effect of pH, temperature, and concentration of enzyme, substrate, product, salt ions, and buffers on the kinetic behavior of urease was examined. A kinetic model of a modified Michaelis-Menten form, incorporating substrate and product inhibition, pH dependence, and temperature effect, was developed to describe the reaction rate. Experimental data indicated that urease in a buffer-free solution was less susceptible to the inhibition of substrate product. The Michaelis constant keeps almost constant with the variation of pH and temperature, and increases with the addition of buffers and salts. The data also suggested that the noncompetitive pattern of the product inhibition, which is not significantly affected by temperature, increases gently with increasing pH. A Monod form rate expression was proposed to analyze the pH effect on the maximum rate. The proposed kinetic model was also examined by the long-time experiments in which pH, substrate, and product concentration varied obviously during the reaction course.     

阳离子表面活性剂溴代十六烷基吡啶胶束对2,4-二硝基氯苯水解反应的影响

阳离子表面活性剂溴代十六烷基吡啶(CPB)胶束对水中OH^-和2, 4-二硝基氯苯的反应有催化作用.随CPB浓度的增大, 反应速率常数开始时迅速增大, 然后趋于常数.OH^-浓度的增大使胶束催化水解的二级速率常数减小.CPB胶束对此反应的速率提高作用比十六烷基三甲基溴化扶胶束更有效.对这两种胶束催化作用的差异进行了讨论.     

Simulaaneous ethanol and cellobiose inhibition of cellulose hydrolysis studied with integrated equations assuming constant or variable substrate concentration

The integrated forms of the Michaelis-Menten equation assuming variable substrate (depletion) or constant substrate concentration were used to study the effect of the simultaneous presence of two exoglucanase Cel7A inhibitors (cellobiose and ethanol) on the kinetics of cellulose hydrolysis. The kinetic parameters obtained, assuming constant substrate (K _m =21 mM, K _ic =0.035 mM; K _icl =1.5×10¹⁵mM; k _cat=12 h⁻¹) or assuming variable substrate (K _m =16 mM, K _ic =0.037 mM; K _icl =5.8×10¹⁴ mM; k _cat=9 h⁻¹), showed a good similarity between these two alternative methodologies and pointed out that bothethanol and cellobiose are competitive inhibitors. Nevertheless, ethanol is a very weak inhibitor, as shown by the large value estimated for the kinetic constant K _icl . In addition, assuming different concentrations of initial accessible substrate present in the reaction, both inhibition and velocity constants are at the same order of magnitude, which is consistent with the obtained values. The possibility of using this kind of methodology to determine kinetic constants in general kinetic studies is discussed, and several integrated equations of different Michaelis-Menten kinetic models are presented. Also examined is the possibility of determining inhibition constants without knowledge of the true accessible substrate concentration.     

Ester hydrolysis by a cyclodextrin dimer catalyst with a metallophenanthroline linking group

A novel beta-cyclodextrin dimer, 1,10-phenanthroline-2,9-dimethyl-bridged-bis(6-monoammonio-beta-cyclodextrin) (phenBisCD, L), was synthesized. Its zinc complex (ZnL) has been prepared, characterized, and applied as a new catalyst for diester hydrolysis. The formation constant (logK(ML)=9.56+/-0.01) of the complex and deprotonation constant (pK(a)=8.18+/-0.04) of the coordinated water molecule were determined by a potentiometric pH titration at (298+/-0.1) K. Hydrolytic kinetics of carboxylic acid esters were performed with bis(4-nitrophenyl) carbonate (BNPC) and 4-nitrophenyl acetate (NA) as substrates. The obtained hydrolysis rate constants showed that ZnL has a very high rate of catalysis for BNPC hydrolysis, giving a 3.89x10(4)-fold rate enhancement over uncatalyzed hydrolysis at pH 7.01, relative to only a 42-fold rate enhancement for NA hydrolysis. Moreover, the hydrolysis second-order rate constants of both BNPC and NA greatly increases with pH. Hydrolytic kinetics of a phosphate diester catalyzed by ZnL was also investigated by using bis(4-nitrophenyl) phosphate (BNPP) as the substrate. The pH dependence of the BNPP cleavage in aqueous buffer shows a sigmoidal curve with an inflection point around pH 8.11, which was nearly identical to the pK(a) value from the potentiometric titration. The k(cat) of BNPP hydrolysis promoted by ZnL was found to be 9.9x10(-4) M(-1) s(-1), which is comparatively higher than most other reported Zn(II)-based systems. The possible intermediate for the hydrolysis of BNPP, BNPC, and NA catalyzed by ZnL is proposed on the basis of kinetic and thermodynamic analysis.     

First kinetic determination of partition coefficients for organic compounds between the three microenvironments of AOT-based microemulsions.

Kinetic data for the hydrolysis of N-picolinoylimidazole (I) and 2,4-dinitrophenylpicolinate (II) in AOT [bis(2-ethylhexyl) sodium sulfosuccinate] microemulsions are used to determine for the first time the two partition constants for each substrate (i.e. K(wi) and K(oi), corresponding to the incorporation of substrate molecules from water microdroplets and the continuous medium, respectively, into the interface). Application of the pseudophase formalism to the partition constants allowed the rate constant in each phase to be determined. The rate of hydrolysis of II increased with decreasing polarity of the medium; as a result, the hydrolysis reaction took place largely at the interface. On the other hand, the rate of hydrolysis of acylimidazole I decreased with decreasing polarity, possibly as a result of changes in the resonance structures of the reagent causing the hydrolysis process to occur preferentially in water microdroplets.     

Homogeneous redox catalysis of multielectron electrochemical reactions: Part II. Competition between homogeneous electron transfer and addition on the catalyst

Homogeneous redox catalytic processes in which catalysis competes with partial destruction of the catalyst are investigated. The kinetics are shown to depend upon three parameters: the excess factor (concentration of substrate over concentration of the catalyst), a dimensionless kinetic parameter representing the rate of the initial homogeneous electron transfer step and a dimensionless parameter representing the competition between the second electron transfer and the addition on the catalyst. Procedures are described that allow the rate constant of the initial electron transfer step and the ratio of the rate constants of the second electron transfer vs. the addition step to be extracted from the experimental data. The reduction of n-butyl chloride and bromide, mediated by aromatic anion radicals, is taken as an example illustrating the application of the proposed procedures to experimental systems.     

A novel method to determine the molecular weight of the oligomers of biodegradable polymer

The biodegradable polymers used in controlled release applications are primarily insoluble polymers which undergo chemical or enzymatic hydrolysis to form soluble monomeric or oligomeric units. To determine the endurance of the polymer after implantation or injection into the body it is important to determine the rate of elimination of the intermediates formed upon the hydrolysis of the polymer. It is essential to identify these intermediates before its elimination rate can be determined. Identification of these intermediates is a problem because they are difficult to isolate, and are thermally and hydrolytically unstable. A new technique is proposed, taking advantage of the neighboring group effect on the hydrolysis of ionic and non-ionic oligomers, to determine the molecular weight of unknown species that are formed upon hydrolysis of the polymer. The technique involves the determination of the specific acid and base catalytic rate constants. To demonstrate the technique, a time sequence synthesis was carried out to synthesize several oligomers that would be forming upon the hydrolysis of poly(butylene tartrate). Hydrolysis studies were conducted with these oligomers and the samples were analyzed by liquid chromtography. The observed rate constant for the hydrolysis under acidic and alkaline pH medium were calculated from the terminal slopes of the first order plot. The observed rate constants were further utilized to calculate the specific acid and base catalytic rate constants. These specific rate constants, along with the micro-hydrogen and -hydroxyl ion catalytic rate constants, were used to determine the molecular weight of the intermediate species. The molecular weight obtained from the kinetic parameters was in excellent agreement with the results obtained from fast atom bombardment mass spectrometry. The same type of analysis can be extended to any multifunctional group compound which has repeat units and can undergo a specific reaction which can be accurately measured.     

New insights on the mechanism of palladium-catalyzed hydrolysis of sodium borohydride from 11B NMR measurements

To gain insight on the mechanistic aspects of the palladium-catalyzed hydrolysis of NaBH(4) in alkaline media, the kinetics of the reaction has been investigated by (11)B NMR (nuclear magnetic resonance) measurements taken at different times during the reaction course. Working with BH(4)(-) concentration in the range 0.05-0.1 M and with a [substrate]/[catalyst] molar ratio of 0.03-0.11, hydrolysis has been found to follow a first-order kinetic dependence from concentration of both the substrate and the catalyst (Pd/C 10 wt %). We followed the reaction of NaBH(4) and its perdeuterated analogue NaBD(4) in H(2)O, in D(2)O and H(2)O/D(2)O mixtures. When the process was carried out in D(2)O, deuterium incorporation in BH(4)(-) afforded BH(4)(-)(n)D(n)(-) (n = 1, 2, 3, 4) species, and a competition between hydrolysis and hydrogen/deuterium exchange processes was observed. By fitting the kinetics NMR data by nonlinear least-squares regression techniques, the rate constants of the elementary steps involved in the palladium-catalyzed borohydride hydrolysis have been evaluated. Such a regression analysis was performed on a reaction scheme wherein the starting reactant BH(4)(-) is allowed both to reversibly exchange hydrogen with deuterium atoms of D(2)O and to irreversibly hydrolyze into borohydroxy species B(OD)(4)(-). In contrast to acid-catalyzed hydrolysis of sodium borohydride, our results indicate that in the palladium-catalyzed process the rate constants of the exchange processes are higher than those of the corresponding hydrolysis reactions.     

Efficient rate enhancement due to intramolecular general base (IGB) assistance in the hydrolysis of N-(o-hydroxyphenyl)phthalimide

The rates of the hydrolyses of N-(o-hydroxyphenyl)phthalimide (1) and N-(o-methoxyphenyl)phthalimide (2), studied at different pH, show that the hydrolysis of 1 involves intramolecular general base (IGB) assistance where the o-O- group of ionized 1 acts as IGB and H2O as the reactant. The rate enhancement due to the IGB-assisted reaction of H2O with ionized 1 is>8x10(4)-fold. Pseudo-first-order rate constant for the reaction of water with 2 is approximately 2x10(3)-fold smaller than the first-order rate constant (0.10 s-1) for pH-independent hydrolysis of 1 within the pH range of 9.60-10.10. Second-order rate constants (kOH) for hydroxide ion-assisted hydrolysis of ionized 1 and 2 are 3.0 and 29.1 M-1 s-1, respectively. The solvent deuterium kinetic isotope effect (dKIE) on the rate of alkaline hydrolysis of 1 and 2 reveals that the respective values of kOH/kOD are 0.84 and 0.78, where kOD represents the second-order rate constant for DO--assisted cleavage of these imides (1 and 2). The value of kwH2O/kdD2O is 2.04, with kwH2O and kdD2O representing pseudo-first-order rate constants for the reactions of ionized 1 with H2O and D2O, respectively.     

Stationary Kinetics of Light Olefin Oxidation by Hydrogen Peroxide in Aqueous Alkali Solutions in the Presence of Fe(III) Oxide

The steady-state kinetics of ethylene and propylene oxidation by hydrogen peroxide in the presence of Fe(III) oxide in aqueous solutions with the permanent adding of H₂O₂ to the reaction medium was studied. The use of an original method for the study of the steady-state reaction kinetics with gas chromatographic detection of substrate consumption from the gas phase made it possible to estimate the apparent rate constants of ethylene oxidation, the ratio of the rate constants of propylene and ethylene oxidation, the reaction orders with respect to the substrate and oxidant concentration, the dependence of the apparent rate constant of ethylene oxidation on the catalyst weight and on the pH of solution, and the apparent activation energy of the process under condition of substrate distribution between the gas and liquid phases. It was found that the kinetic isotope effect in ethylene oxidation is almost absent when completely deuterated ethylene is used.     

Base catalyzed hydrolysis of Aerosol OT in Aqueous and Aquo-Dioxane media

The OH^? ion catalyzed hydrolysis of AOT and sodium mono-methyl succinate in aqueous and aquo-dioxane media has been studied. The second-order rate constant for the former has been found to be nearly ten times slower than that of the latter. At AOT concentrations above CMC, the rate constants become insensitive to the concentration in the studied range of temperature, 25°C–40°C. The activation parameters for the kinetic process have been found to be reasonable, negative entropy of activation has supported a stable transition state complex. A good enthalpy-entropy compensation of the kinetic process has been found both for AOT and sodium monomethyl succinate in aqueous and aquo-dioxane media supporting similar nature of the transition state complexes in the hydrolytic reactions. The enthalpies of hydrolysis of AOT and the half ester of succinic acid in aqueous and aquo-dioxane media have been also reported. © 1994 John Wiley & Sons, Inc.     

Hydrolysis of p-nitrophenyl picolinate catalyzed by metal complexes of N-alkyl-3,5-bis(hydroxymethyl)-1,2,4-triazole in CTAB micelles

Two lipophilic ligands containing triazole and hydroxyl groups, N-alkyl(C(n)H(2n+1))-3,5-bis(hydroxymethyl)-1,2,4-triazole (n=10 and 12), were synthesized. Effects of their Cu(II) and Ni(II) complexes on the hydrolysis of p-nitrophenyl picolinate (PNPP) in cetyltrimethylammonium bromide (CTAB) micelles have been investigated kinetically, and some kinetic parameters of the reactions were obtained by employing the ternary complex kinetic model for metallomicellar catalysis. It was found that Cu(II) complexes of these triazole-based ligands showed more effective catalytic activity on the hydrolysis of PNPP than Ni(II) complexes. Also, the apparent first-order rate constants for product formation in the metallomicellar phase (k(N)(')), the association constants between the substrate and the binary complex (K(T)), and the association constants between the metal ion and the ligand (K(M)) increased with an increase in pH value, which may be attributed to an increase in the nucleophilicity of the hydroxyl groups in the ligand or the electrophilicity of the substrate at higher pH. In addition, at constant pH, k(N)(') and K(T) increased with an increase in the hydrocarbon chain length of the ligand, while K(M) decreased.     

Cytosol-mimetic chemistry: kinetics of the trypsin-catalyzed hydrolysis of p-nitrophenyl acetate upon addition of polyethylene glycol and N-tert-butyl acetoacetamide

The sensitivity of an enzyme to its environment has provoked much interest both for its immediate relevance to biochemistry and for the use of enzymes in chemical synthesis. The intercellular or extracellular environment in which an enzyme naturally operates is crowded with macromolecular, small-molecule, and ionic solutes and hence is markedly different from the dilute aqueous buffer solutions commonly cited for comparisons of biochemical processes. We report the results of a kinetic study into the effects of such a crowded solution on the rate of an enzyme-mediated process-the trypsin-catalyzed hydrolysis of a nonnatural substrate ester. The catalytic rate constant decreases linearly with solvent polarity, but substrate binding is independent of the concentration of added crowding agent up to 395 g/L.     

Kinetics of N-glutaryl-L-phenylalanine p-nitroanilide hydrolysis catalyzed by alpha-chymotrypsin in aqueous solutions of alkyltrimethylammonium bromides

The rate of N-glutaryl-L-phenylalanine p-nitroanilide hydrolysis catalyzed by alpha-chymotripsin has been measured in aqueous solutions of cetyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, and dodecyltrimethylammonium bromide at concentrations below and above their critical micellar concentrations (CMC). For the three surfactants considered superactivity was observed, with maximum catalytic efficiencies taking place near the corresponding CMCs. The effect of the surfactants after the CMCs is mostly due to a decreased thermodynamic activity of the substrate due to its incorporation into the micelles. After addition of the surfactants, the Michaelis constant values (corrected to take into account the free substrate concentration) tend to decrease, passing through an ill defined minimum, afterwards reaching a constant value. The catalytic rate constants show the same profiles that the catalytic efficiency, being maxima near the surfactants CMCs. This maximum is more important for the surfactant having the shorter tail. This result is explained by considering that the hydrophobicity of the surfactant influences more the CMC than its association to the enzyme.     

Kinetic evidence for hydrophobically stabilized encounter complexes formed by hydrophobic esters in aqueous solutions containing monohydric alcohols.

The pH-independent hydrolysis of four esters, p-methoxyphenyl 2,2-dichloroethanoate (1a), p-methoxyphenyl 2,2-dichloropropanoate (1b), p-methoxyphenyl 2,2-dichlorobutanoate (1c), and p-methoxyphenyl 2,2-dichloropentanoate (1d), in dilute aqueous solution has been studied as a function of the molality of added cosolutes ethanol, 1-propanol, and 1-butanol. The rate constants for the neutral hydrolysis decrease with increasing cosolute concentration. These kinetic medium effects respond to both the hydrophobicity of the ester and of the monohydric alcohol. The observed rate effects were analyzed using both a thermodynamic and a kinetic model. The kinetic model suggests a molecular picture of a hydrophobically stabilized encounter complex, with equilibrium constants K(ec) often smaller than unity, in which the cosolute blocks the reaction center of the hydrolytic ester for attack by water. The formation of these encounter complexes leads to a dominant initial-state stabilization as follows from the thermodynamic model. Decreases in both apparent enthalpies and entropies of activation for these hydrolysis reactions correspond to unfavorable enthalpies and favorable entropies of complexation, which confirms that the encounter complexes are stabilized by hydrophobic interactions.     

Micellization and catalytic activity of the cetyltrimethylammonium bromide-Brij 97-water mixed micellar system

Surface tension measurements and the kinetic study of the basic hydrolysis of ethyl p-nitrophenyl chloromethyl phosphonate were used to examine the structural behavior and catalytic activity of the cethyltrimethylammonium bromide (CTAB)-polyoxyethylene (10) oleyl ether, C(18)H(35)(OCH(2)CH(2))(10)OH (Brij 97)-water mixed micellar system. Application of the regular solution model to the experimental data yields the value of the interaction parameter beta as -4.6, which indicates an attractive interaction of the surfactants in the mixed micelle and reflects synergistic solution behavior of the mixture. The mixed micellar composition is found to be enriched in the surfactant with the lower critical micelle concentration (cmc). In the kinetic study a nonmonotonic change in the pseudo-first-order rate constant of basic hydrolysis of the substrate is observed with increasing mole fraction of nonionic surfactant. The pseudophase micellar model reveals that the concentration factor mainly contributes to the catalytic effect, while the microenvironmental factor plays a negative role.     

Mechanism of 1,4,5,8-naphthalene tetracarboxylic acid dianhydride hydrolysis and formation in aqueous solution

The study of highly conjugated, carbonyl-containing molecules such as 1,4,5,8-naphthalene tetracarboxylic dianhydride, III, is of interest since reactivity differences and transmission of electronic effects through the conjugated framework can be evidenced. The kinetics of hydrolysis of III in aqueous solution were determined from 5 M acid to pH 10. In basic solution hydrolysis of III yields, sequentially, 1,4,5,8-naphthalene diacid monoanhydride, II, and 1,4,5,8-naphthalene tetracarboxylic acid, I. The second order rate constant for alkaline hydrolysis is 200 fold higher for the first ring opening. The water-catalyzed hydrolysis of III yields a pH-dependent mixture of ionic forms of I and II. The rate constant for water-catalyzed hydrolysis of III is 25 fold higher than that for II. In concentrated acid the rates for reaching equilibrium (I, II and III) increase and III is the major product. The pK(a)s of I (3.24, 5.13 and 6.25) and II (3.05, 5.90) were determined by potentiometric, fluorescence and UV spectroscopy titrations and by quantitative fit of the kinetic and equilibrium data. The apparent, pH-dependent, equilibrium constants, K(EqII), for anhydride formation between I and II were obtained from the UV spectra. The quantitative fit of kinetic and equilibrium data are consistent with the assumption that anhydride formation only proceeds with the fully protonated species for both I and II and permitted the estimation of the equilibrium constants for anhydride formation, K(EqII). The value of K(EqII) (I <==> II) between pH 1 and 6 was ca. 5. Geometry optimization calculations in the gas phase of the reactions of III in alkaline, neutral and acid conditions, at the DFT level of theory, gave electronic distributions that were qualitatively consistent with the experimental results.