Modulation of tautomeric equilibria by ionic clusters. Acetylacetone in solutions of lithium perchlorate-diethyl ether.

Acetylacetone (2,4-pentanedione, 1) is a molecule whose tautomeric forms are in dynamic equilibrium. Concentrated salt solutions in nonaqueous solvents exert a remarkable influence on the keto-enol ratio of this beta-diketone. The keto content of 1 increases from 5% in pure diethyl ether to 84.5% in a 4.14 M lithium perchlorate-diethyl ether (LPDE) solution, a nearly 17-fold increase. The equilibrium expression, K = [keto]/[enol] = k(f)/k(r), exhibits a linear dependence on [LiClO(4)], with the formal order of participation of lithium ion in the equilibrium being 1.0. A kinetic analysis reveals that k(f) is independent of LPDE concentration, whereas k(r) displays an inverse dependence on salt concentration, indicating preferential coordination of the keto tautomer with Li(+). Although 1 exits as the enol in water only to the extent of 16%, the addition of lithium perchlorate further reduces this figure. In an aqueous 4.02 M LiClO(4) solution, acetylacetone enol accounts for only 4.6% of the total amount of 2,4-pentanedione present. It has also been found that acetylacetone itself is an excellent solvent for LiClO(4) as well as for NaClO(4) with solutions containing up to 7.5 M LiClO(4) attainable. The enol content of 1 decreases dramatically from 81% to 7.4% on going from the neat liquid to a solution of 6.39 M LiClO(4) in acetylacetone.

Electrostatic modulation by ionic aggregates: charge transfer transitions in solutions of lithium perchlorate-diethyl ether.

The ionic environment within solutions of lithium perchlorate-diethyl ether (LPDE) was probed by utilizing the extraordinary spectral shifts these media impart on various nitroanilines at 25 degrees C. These compounds all have UV-visible spectra that are sensitive to the polarity of the medium and the nitroanilines investigated all exhibited varying degrees of solvatochromatic behavior in LPDE solutions. In all cases, the low-energy absorbance band exhibited a dependence upon LiClO(4) concentration throughout the entire solubility range investigated. For 4-nitroaniline and N,N-dimethyl-4-nitroaniline bathochromic shifts of 51.3 and 62.0 nm, respectively, were observed on going from pure ether to a 5.7 M LPDE solution, corresponding to a stabilization of 10.55 and 11.13 kcal mol(-1), respectively, for this transition. Thus, as the medium changes from diethyl ether to one containing ionic clusters of lithium perchlorate-diethyl ether, less energy is required to transfer the molecules from their ground states to their first excited states. For 2,6-dibromo- and 2,6-diiodo-4-nitroaniline smaller red shifts of 19.0 and 9.0 nm, respectively, were noted over the same concentration range of LPDE, resulting in stabilizations of 4.45 and 2.11 kcal mol(-1), respectively. Analysis of the observed molar transition energies indicates that for 4-nitroaniline and N,N-dimethyl-4-nitroaniline the stabilization of the zwitterionic excited states of such push-pull molecules is on the order of 2.0 kcal mol(-1) per mol of added salt. Furthermore, such stabilization is independent of the composition of the media. Thus these compounds can act as solvent polarity indicators for LPDE solutions throughout the entire solubility range of LiClO(4) in diethyl ether. As such, linear relationships are seen between the E(T) values of 4-nitroaniline and N,N-dimethyl-4-nitroaniline and the log of the second-order rate constants for the [4+2] cycloaddition reaction of 9,10-dimethylanthracene and acrylonitrile in LPDE. We also observe linear relationships between the E(T) values of 4-nitroaniline and N,N-dimethyl-4-nitroaniline and the keto-enol ratio of acetylacetone in LPDE.

Ternary complexes of liver alcohol dehydrogenase.

Liver alcohol dehydrogenase (LADH; E.C. 1.1.1.1) provides an excellent system for probing the role of binding interactions with NAD(+) and alcohols as well as with NADH and the corresponding aldehydes. The enzyme catalyzes the transfer of hydride ion from an alcohol substrate to the NAD(+) cofactor, yielding the corresponding aldehyde and the reduced cofactor, NADH. The enzyme is also an excellent catalyst for the reverse reaction. X-ray crystallography has shown that the NAD(+) binds in an extended conformation with a distance of 15 A between the buried reacting carbon of the nicotinamide ring and the adenine ring near the surface of the horse liver enzyme. A major criticism of X-ray crystallographic studies of enzymes is that they do not provide dynamic information. Such data provide time-averaged and space-averaged models. Significantly, entries in the protein data bank contain both coordinates as well as temperature factors. However, enzyme function involves both dynamics and motion. The motions can be as large as a domain closure such as observed with liver alcohol dehydrogenase or as small as the vibrations of certain atoms in the active site where reactions take place. Ternary complexes produced during the reaction of the enzyme binary entity, E-NAD(+), with retinol (vitamin A alcohol) lead to retinal (vitamin A aldehyde) release and the enzyme binary entity E-NADH. Retinal is further metabolized via the E-NAD(+)-retinal ternary complex to retinoic acid (vitamin A acid). To unravel the mechanistic aspects of these transformations, the kinetics and energetics of interconversion between various ternary complexes are characterized. Proton transfers along hydrogen bond bridges and NADH hydride transfers along hydrophobic entities are considered in some detail. Secondary kinetic isotope effects with retinol are not particularly large with the wild-type form of alcohol dehydrogenase from horse liver. We analyze alcohol dehydrogenase catalysis through a re-examination of the reaction coordinates. The ground states of the binary and ternary complexes are shown to be related to the corresponding transition states through topology and free energy acting along the reaction path.

Bioinorganic and bioorganic studies of liver alcohol dehydrogenase.

Liver alcohol dehydrogenase (E.C.1.1.1.1) is an NAD(+)/NADH dependent enzyme with a broad substrate specificity being active on an assortment of primary and secondary alcohols. It catalyzes the reversible oxidation of a wide variety of alcohols to the corresponding aldehydes and ketones as well as the oxidation of certain aldehydes to their related carboxylic acids. Although the bioinorganic and bioorganic aspects of the enzymatic mechanism, as well as the structures of various ternary complexes, have been extensively studied, the kinetic significance of certain intermediates has not been fully evaluated. Nevertheless, the availability of computer-assisted programs for kinetic simulation and molecular modeling make it possible to describe the biochemical mechanism more completely. Although the true physiological substrates of this zinc metalloenzyme are unknown, alcohol dehydrogenase effectively catalyzes not only the interconversion of all-trans-retinol and all-trans-retinal but also the oxidation of all-trans-retinal to the corresponding retinoic acid. Retinal and related vitamin A derivatives play fundamental roles in many physiological processes, most notably the vision process. Furthermore, retinoic acid is used in dermatology as well as in the prevention and treatment of different types of cancer. The enzyme-NAD(+)-retinol complex has an apparent pK(a) value of 7.2 and loses a proton rapidly. Proton inventory modeling suggests that the transition state for the hydride transfer step has a partial negative charge on the oxygen of retinoxide. Spectral evidence for an intermediate such as E-NAD(+)-retinoxide was obtained with enzyme that has cobalt(II) substituted for the active site zinc(II). Biophysical considerations of water in these biological processes coupled with the inverse solvent isotope effect lead to the conclusion that the zinc-bound alkoxide makes a strong hydrogen bond with the hydroxyl group of Ser48 and is thus activated for hydride transfer. Moderate pressure accelerates enzyme action indicative of a negative volume of activation. The data with retinol is discussed in terms of enzyme stability, mechanism, adaptation to extreme conditions, as well as water affinities of substrates and inhibitors. Our data concern all-trans, 9-cis, 11-cis, and 13-cis retinols as well as the corresponding retinals. In all cases the enzyme utilizes an approximately ordered mechanism for retinol-retinal interconversion and for retinal-retinoic acid transformation.

Water in enzyme reactions: biophysical aspects of hydration-dehydration processes.

Water has been recognized as one of the major structuring factors in biological macromolecules. Indeed, water clusters influence many aspects of biological function, and the water-protein interaction has long been recognized as a major determinant of chain folding, conformational stability, internal dynamics, binding specificity and catalysis. I discuss here several themes arising from recent progress in understanding structural aspects of 'direct' and 'indirect' ligands in terms of enzyme-substrate interactions, and the role of water bridges in enzyme catalysis. The review also attempts to illuminate issues relating to efficiency, through solvent interactions associated with enzymic specificity, and versatility. Over the years, carbonic anhydrase (CA; carbonate hydrolyase, EC 4.2.1.1) has played a significant role in the continuing delineation of principles underlying the role of water in enzyme reactions. As a result of its pronounced catalytic power and robust constitution CA was transformed into a veritable 'laboratory' in which active site mechanisms were rigorously tested and explored.

The inhibition of bovine carbonic anhydrase by saccharin and 2- and 4-carbobenzoxybenzene sulfonamide.

The present work demonstrates that the high-activity zinc metalloenzyme, carbonic anhydrase (CA II) from bovine erythrocytes is inhibited by the cyclic sulfimide, saccharin, and 2- and 4-carbobenzoxybenzene sulfonamide. A spectrophotometric method was employed to monitor the enzymatically catalyzed hydrolysis of p-nitrophenyl acetate by following the increase in absorbance at 410 nm which accompanies p-nitrophenoxide/p-nitrophenol formation. The more rapid enzymatic hydration of CO2 was monitored by using a stopped-flow spectrophotometer as well as by a modified colorimetric method of Wilbur and Anderson. The studies show that, at a given molar ratio of inhibitor to enzyme, the degree of inhibition of the enzymaic hydration of CO2 and hydrolysis of p-nitrophenyl acetate by the inhibitory compounds is essentially the same. Kinetic analyses were made at 25.0 degrees at pH 6.5 (MES buffers), pH 6.9 (HEPES buffers) and pH 7.9 (HEPES buffers) with ionic strength regulated by the addition of appropriate quantities of sodium sulfate. Lineweaver-Burk plots were used to evaluate apparent inhibition constants for each of the three inhibitors. For all the inhibitors studied, inhibition appears to be mixed (competitive/noncompetitive). For saccharin in the presence of sodium sulfate, the extent of inhibition is considerably decreased. It was found for the three inhibitors that the inhibitory potency decreases with increasing pH, and that the inhibitory potency is extremely sensitive to the shape of these rather closely related molecules. For example, apparent inhibition constants for the enzymatic hydrolysis of p-nitrophenyl acetate at pH 6.9 were Ki (saccharin) = 0.20 mM, Ki (2-carbobenzoxybenzene sulfonamide) = 0.54 mM and Ki (4-carbobenzoxybenzene sulfonamide) = 1.6 microM. For the enzymatic hydration of CO2 at pH 6.9, 0.10 mM saccharin caused 50% inhibition while 7.0 nM 4-carbobenzoxybenzene sulfonamide resulted in 50% inhibition. The results suggest that sulfonamide inhibition is caused by formation of a monodentate ligand at the zinc ion of the enzyme active site and that the more linear 4-carbobenzoxybenzene sulfonamide is better able to enter a conical enzyme active site than is 2-carbobenzoxybenzene sulfonamide or saccharin.

Bistability and the ordered bimolecular mechanism.

An equation describing the instantaneous velocity of an ordered bimolecular enzymatic reaction that exhibits inhibition by substrate and product was derived. Using kinetic constant values for horse liver alcohol dehydrogenase, the velocity expression was applied to an open-reaction system. The calculated steady-state surfaces displayed regions of bistability, which further substantiates the link between substrate inhibition and multiple steady states. This general computational approach may be applied to any system that can be described by an instantaneous velocity equation.

Kinetics and mechanism of methanol and formaldehyde interconversion and formaldehyde oxidation catalyzed by liver alcohol dehydrogenase.

It has been shown that the hydrophobic interaction in the active-site plays a fundamental role in substrate binding. Proper molecular orientation is required for hydride transfer (Dalziel and Dickinson, 1967). For methanol, the binding is unfavored due to the lack of a hydrophobic chain. In the enzyme-coenzyme-substrate complex, the small methyl group of the substrate is not held in a fixed position, resulting in a low hydride transfer rate. The binding of NAD+ to the enzyme does not exhibit a significant effect on the binding of methanol, nor does methanol affect NAD+ binding. In the presence of LADH, methanol is oxidized by NAD+ to formaldehyde, while formaldehyde can be oxidized by NAD+ to formate ion or reduced by NADH to methanol. These reactions follow a rapid equilibrium random mechanism. Among these three reactions, the reduction of formaldehyde is the most rapid. The rate of formaldehyde oxidation is faster than the oxidation of methanol. Our study with these non-hydrophobic substrates provides an important bridge between the bioinorganic activation of zinc-bound water and the bioorganic oxidation of ethanol. Furthermore, it furnishes some insight into an enzymatic system that is so highly sensitive to small changes in substrate chain length that it can magnify the consequence of a modest change in substrate hydrophobicity.

Zinc-activated alcohols in ternary complexes of liver alcohol dehydrogenase.

Activation parameters for each reaction step in the kinetic mechanism of liver alcohol dehydrogenase have been measured for the oxidation of ethanol and the reduction of acetaldehyde. In the oxidation process, the highest enthalpy of activation, 9.7 kcal/mol, occurs for the turnover of the liver alcohol dehydrogenase-NAD(+)-ethanol ternary complex. To investigate if this enthalpy requirement represents a change in the ionization state of ethanol bound in the ternary complex, inhibition of ethanol oxidation was determined using the following series of small, electronegative alcohols with pKa values ranging from 12.37 to 15.5: 2,2,2-trifluoroethanol, 2,2,2-trichloroethanol, 2,2,2-tribromoethanol, 2,2-dichloroethanol, 2,2-difluoroethanol, propargyl alcohol, 3-hydroxypropionitrile, 2-chloroethanol, 2-iodoethanol, 2-methoxyethanol, ethylene glycol, and methanol. The observed inhibition patterns were analyzed according to several kinetic inhibition models; in each case, the best fit model was used to determine the substrate competitive inhibition constant. A plot of the logarithm of these inhibition constants is shown to be dependent on the pKa values of the inhibiting alcohols with a slope approaching -1, indicating that inhibition is controlled by a proton loss from the alcohol. The observed competitive inhibition behavior, coupled with crystallographic studies depicting a direct ligation of an alcohol oxygen to the catalytic zinc ion, indicates that inhibition is controlled by the formation of a zinc-bound alkoxide. Because the inhibiting alcohols are structurally homologous to ethanol, a relationship between the inhibition constant and the inhibiting alcohol's pKa can be derived to show that the pKa of an alcohol bound in a ternary complex is also dependent on its pKa as a free alcohol. Ternary complex pKa values have been determined for ethanol and the inhibiting alcohols.

Origin of viscosity effects in carbonic anhydrase catalysis. Kinetic studies with bulky buffers at limiting concentrations.

In our earlier paper we showed that the rates of CO2 hydration and HCO3- dehydration catalyzed by the high-activity form of mammalian erythrocyte carbonic anhydrase (CA II) were dependent on solution viscosity increase and that the effect was linked to some kind of proton-transfer-related event [Pocker, Y., & Janjic, N. (1987) Biochemistry 26, 2597-2606]. In order to further elucidate the source of the observed viscosity effect, the dependence of kcat and Km for CA II catalyzed HCO3- dehydration at pH 5.90 on sucrose-induced viscosity increase was investigated at several concentrations of 2-(N-morpholino)ethanesulfonic acid (MES) buffer, including the very low buffer concentration region (less than 10 mM) where the proton transfer between the shuttle group on the enzyme and buffer becomes rate limiting. In all examined cases, kcat steadily decreased with added sucrose while Km remained independent of the viscosity increase. The extent to which this reaction was dependent on viscosity was found to be constant, within experimental error, over the entire range of MES buffer concentrations studied (1-20 mM). Furthermore, the viscosity effect was qualitatively and quantitatively the same when an exceptionally large buffer (i.e., bovine serum albumin) was used instead of the more commonly used biological buffer (i.e., MES).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential modification of specificity in carbonic anhydrase catalysis.

Incubation of carbonic anhydrase II with acrolein results in a rapid, time-dependent loss of all but approximately 3-6% of the original catalytic activity toward CO2 hydration and HCO3- dehydration, with the inactivation rate being first-order in both acrolein and the enzyme. The pH dependence of the inactivation rate constant can be adequately described with a function incorporating a pK alpha of 7.15 and a maximal value for kinact [corrected] of 26.2 M-1 min-1, indicating that at least one of the catalytically essential residues that ionizes at this pH is involved in the modification scheme. The amount of residual CO2 hydratase activity is proportional to the molar excess of acrolein over carbonic anhydrase II with 5 histidyl and 3 lysyl residues being subject to alkylation under conditions where [acrolein] to [carbonic anhydrase II] ratio is greater than 100. Because all lysyl residues were shown previously to be amidinated without detectable loss of activity, it was assumed that the modification of one (or more) of the histidines was primarily responsible for the observed inactivation. The number of modified histidyl residues could be related to residual activity by using the statistical analysis of Tsou (Tsou, C.-L. (1962) Sci. Sin. (Engl. Ed.) 11, 1535-1558) which indicates that one essential histidine reacts approximately four times faster than the other (histidyl) residues. In sharp contrast with the phenomenon observed in connection with CO2 hydration and HCO3- dehydration, acrolein improves the catalytic efficiency of the enzyme toward p-nitrophenyl acetate hydrolysis and acetaldehyde hydration, with the relative activity increasing by approximately 12 and 34%, respectively. The widely differing effects imparted by the same reagent represent the first step toward differential control of the specificity of carbonic anhydrase II.

Molecular basis of ionic strength effects: interaction of enzyme and sulfate ion in CO2 hydration and HCO3- dehydration reactions catalyzed by carbonic anhydrase II.

CO2 hydration and HCO3- dehydration reactions catalyzed by carbonic anhydrase II have been examined at various concentrations of sodium sulfate with a stopped-flow technique. We find that at low ionic strength CO2 hydration and HCO3- dehydration reaction rates remain unaffected by varying the salt concentration at pH higher than 7.0, while the reaction rates decrease with increasing ionic strength at low pH. For CO2 hydration reactions, salt effects reside only in the kcat term, not in the Km term, whereas for HCO3- dehydration reactions, salt effects reside only in the Km term, not in the kcat term. In this regime, the salt concentration dependence of the turnover rate for CO2 hydration at low pH is attributed to an electrostatic effect on the ionization constants of the enzyme and/or enzyme-substrate complex, which in turn affect the pH profile of kcat. The rates of the bimolecular interaction between the uncharged CO2 molecule and carbonic anhydrase II at high pH are unaffected by low salt concentration while the rates of the bimolecular interaction of HCO3- with enzyme at low pH decrease with increasing salt concentration, consistent with a negative salt effect on an electrostatically enhanced diffusion of the negatively charged substrate to the positively charged active site. These bimolecular reactions between enzyme and substrate at low ionic strength obey rate equations derived from the Debye-Hückel limiting law and the transition-state theory. Simple linear relationships between the logarithm of the catalytic constant, log kdenz, and the square root of the ionic strength were established.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme kinetics in solvents of increased viscosity. Dynamic aspects of carbonic anhydrase catalysis.

The dependence of enzymatic catalysis on diffusion rates in solution was examined with regard to high specific activity carbonic anhydrase (CA II) by varying the viscosity of the reaction medium with added glycerol, sucrose, and ficoll (a copolymer of sucrose and epichlorohydrin). Responses of the Michaelis-Menten parameters associated with CO2 hydration and HCO3- dehydration were deduced and analyzed by utilizing a spectrophotometric stopped-flow technique. It was found that both kcatHCO3 (= 3.9 X 10(5) s-1 at pH 5.90) and kcatCO2 (= 1.2 X 10(5) s-1 at pH 5.90 and 8.6 X 10(5) s-1 at pH 8.80) steadily decreased with the addition of monomeric viscogen while both KmHC03- (= 20 mM at pH 5.90) and KmCO2 (= 18 mM at pH 5.90 and 13 mM at pH 8.80) remained independent of viscosity, within experimental error. These results indicate that some kind of proton-transfer-related event is primarily responsible for the observed rate decrease. The three polyhydroxy cosolutes exhibited significant differences with regard to the magnitude of the viscosity effect on the kcat of the enzyme, with glycerol affecting the largest decrease, sucrose affecting a moderate one, and ficoll having virtually no effect. The discrepancy between glycerol and sucrose could be largely reconciled by correcting for diffusion-unrelated effects as estimated from rate studies of considerably slower CA II catalyzed acetaldehyde hydration and p-nitrophenyl acetate hydrolysis. Ficoll, however, was found to be unsuitable as a viscogenic probe because it failed to appreciably decrease the mobilities of smaller ions (as deduced from electrolytic conductance measurements) despite its capacity to greatly increase the macroscopic viscosity of the medium. Our best estimates indicate that this reaction comes within ca. 30% of the diffusion limit at 0.890 cP and 25 degrees C for both CO2 hydration and HCO3- dehydration reactions. However, it is reasonable to expect this value to be considerably higher in the natural environment of the enzyme because of the relatively high viscosities attained in the interior of erythrocytes.

Liver alcohol dehydrogenase: metabolic and energetic aspects.

This paper presents a series of experiments that define the energetics of liver alcohol dehydrogenase and allow the construction of a free energy profile for the ethanol-acetaldehyde interconversion. Because of its broad specificity, we have also performed a series of kinetic studies designed to test the metabolic efficiency and the mode of action of this enzyme in regard to the reversible oxidation of Vitamin A alcohol (retinol). The slower oxidation of Vitamin A aldehyde (retinal) to Vitamin A acid, (retinoic acid) was also investigated.