Partitioning of fluorescent phospholipid probes between different bilayer environments. Estimation of the free energy of interlipid hydrogen bonding.

Fluorescence spectroscopy has been used to monitor the partitioning of a series of exchangeable neutral phospholipid probes, labeled with carbazole, indolyl or diphenylhexatrienyl moieties, between large unilamellar vesicles containing 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1,2-dioleoyloxy-3-(trimethylammonio) propane (DOTAP) or N-hexadecyl-N-(9-octadecenyl)-N,N-dimethylammonium chloride (HODMA). Phosphatidylethanolamine (PE) probes desorb from POPC-containing vesicles at markedly slower rates than do phosphatidylcholine (PC) probes with the same acyl chains. The rate of probe desorption from such vesicles is progressively enhanced by successive N-methylations of the amino group but not by methylation of C-2 of the ethanolamine moiety, a modification that leaves unaltered the hydrogen-bonding capacity of the polar headgroup. By contrast, the rates of desorption of different probes (with the same acyl chains) from HODMA or from DOTAP vesicles are much more comparable and reflect no clear systematic influence of the headgroup hydrogen-bonding capacity. Equilibrium-partitioning measurements indicate that the relative affinities of different probes for PC-rich vesicles, in competition with HODMA or DOTAP vesicles, increase with increasing hydrogen-bonding capacity of the probe headgroup in the order PC less than N,N-dimethyl PE less than N-methyl PE less than PE approximately phosphatidyl-2-amino-1-propanol. From such partitioning data, we estimate that interlipid hydrogen-bonding interactions (in competition with lipid-water interactions) contribute roughly -300 cal mol-1 to the free energy of a PE molecule in a hydrated liquid-crystalline phospholipid bilayer; this free-energy contribution is somewhat smaller, but still significant, for N-mono- and dimethylated PE's.     

Roles of substrate distortion and intramolecular hydrogen bonding in enzymatic catalysis by scytalone dehydratase.

Alternative substrates and site-directed mutations of active-site residues are used to probe factors controlling the catalytic efficacy of scytalone dehydratase. In the E1cb-like, syn-elimination reactions catalyzed, efficient catalysis requires distortion of the substrate ring system to facilitate proton abstraction from its C2 methylene and elimination of its C3 hydroxyl group. Theoretical calculations indicate that such distortions are more readily achieved in the substrate 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO) than in the physiological substrates vermelone and scytalone by approximately 2 kcal/mol. A survey of 12 active-site amino acid residues reveals 4 site-directed mutants (H110N, N131A, F53A, and F53L) have higher relative values of k(cat) and k(cat)/K(m) for DDBO over scytalone and for DDBO over vermelone than the wild-type enzyme, thus suggesting substrate-distortion roles for the native residues in catalysis. A structural link for this function is in the modeled enzyme-substrate complex where F53 and H110 are positioned above and below the substrate's C3 hydroxyl group, respectively, for pushing and pulling the leaving group into the axial orientation of a pseudo-boat conformation; N131 hydrogen-bonds to the C8 hydroxyl group at the opposite end of the substrate, serving as a pivot for the actions of F53 and H110. Deshydroxyvermelone lacks the phenolic hydroxyl group and the intramolecular hydrogen bond of vermelone. The relative values of k(cat) (95) and k(cat)/K(m) (1800) for vermelone over deshydroxyvermelone for the wild-type enzyme indicate the importance of the hydroxyl group for substrate recognition and catalysis. Off the enzyme, the much slower rates for the solvolytic dehydration of deshydroxyvermelone and vermelone are similar, thus specifying the importance of the hydroxyl group of vermelone for enzyme catalysis.     

Testing electrostatic complementarity in enzyme catalysis: hydrogen bonding in the ketosteroid isomerase oxyanion hole

A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing p K _a models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50–0.76 ppm/p K _a unit, suggesting a bond shortening of ˜0.02 Å/p K _a unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (ΔΔG = −0.2 kcal/mol/p K _a unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (ΔΔH = −2.0 kcal/mol/p K _a unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of ˜300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution.     

The question of differential hydrogen bonding in the mechanism of catalysis by serine proteases

Two mechanisms recently proposed to account for the action of serine in proteases differ on the matter of hydrogen bonding between enzyme and substrate in the Michaelis complex and acyl enzyme. One mechanism involves the removal of hydrogen bonds as a means of raising the free energy of the Michaelis complex toward that of the transition state and thereby effecting catalysis. This report shows that the kinetic parameters $\text{k}{}_2\text{K}{}_{\mathrm{<mtext>s</mtext>}}$ and k₃ for the α-chymotrypsin-catalyzed hydrolysis of a series of halogen-substituted phenyl acetates respond to electron with-drawal and concomitant changes in hydrogen bond energy in a nearly identical fashion. Reference to thermodynamic studies of hydrogen bonding in free solution shows that these data are not consistent with hydrogen bond removal in the Michaelis complex and acyl enzyme but do support the alternative mechanism, in which hydrogen bonding of substrate is maintained along the entire reaction coordinate.     

Studies on hydrogen bonds. Part V--Hydrogen bonding in energy minimization studies of peptides

An energy term, representing the N-H...O type of hydrogen bond, which is a function of the hydrogen bond length (R) and angle (theta) has been introduced in an energy minimization program, taking into consideration its interpolation with the non-bonded energy for borderline values of R and theta. The details of the mathematical formulation of the derivatives of the hydrogen bond function as applicable to the energy minimization have been given. The minimization technique has been applied to hydrogen bonded two and three linked peptide units (gamma-turns and beta-turns), and having Gly, Ala and Pro side chains. Some of the conformational highlights of the resulting minimum energy conformations are a) the occurrence of the expected 4----1 hydrogen bond in all of the burn-turn tripeptide sequences and b) the presence of an additional 3----1 hydrogen bond in some of the type I and II tripeptides with the hydrogen bonding scheme in such type I beta-turns occurring in a bifurcated form. These and other conformational features have been discussed in the light of experimental evidence and theoretical predictions of other workers.     

Binding of transition metal complexes to guanine and guanine–cytosine: hydrogen bonding and covalent effects

Density functional calculations and Atoms in Molecules analysis are used to investigate the role of covalent and hydrogen bondings in determining the binding of transition metal complexes to guanine, and the subsequent effect on pairing with cytosine. Hydrogen bonding is ubiquitous, and typically contributes ca. 10% to overall binding, a value that varies with the coordination site on guanine, as well as metal and ligands. Early transition metals show a clear preference for the O6 position, while later ones prefer N7, the crossover point coming at the vanadium group. Metallation at N7 causes a redistribution of hydrogen bonding strength between guanine and cytosine, but does not greatly affect the overall pairing energy. In contrast, metallation at O6 strongly reduces the pairing energy, as may be expected given the role of O6 in pairing guanine with cytosine. This effect can be quantified using electron density properties, and seems to be due to both electrostatic repulsion from the positive metal centre and a redistribution of electron density within guanine itself. Qualitative agreement with experimental mass spectroscopic results is obtained.     

Binding affinity and biological activity of oxygen and sulfur isosteres at melatonin receptors as a function of their hydrogen bonding capability

Analogues of melatonin (1) and of N-acetyl 5-ethoxytryptamine (3) in which the oxygen atoms are replaced by sulfur have been prepared and tested against human and amphibian melatonin receptors. All sulfur analogues show a decreased binding affinity at human MT1 and MT2 receptors and a reduced potency as melatonin agonists on the Xenopus melanophore assay. The 5-methoxy oxygen of melatonin is significantly more important for receptor binding than the amide oxygen. N-Acetyl 5-ethoxytryptamine shows a decrease in both binding affinity and potency in comparison with melatonin. In this series, replacing either the ethoxy or amide oxygen by sulfur has a similar but smaller effect on both binding affinity and potency. Using K(B)(H) values from Abraham's equations we have assessed the possibility of estimating EC50 values for sulfur isosteres from the EC50 values of their oxygen analogues.     

PH and growth of Torulopsis pintolopesii in media containing various sugars as carbon and energy sources.

Growth within the pH range 2 to 8 of a strain of the yeast Torulopsis pintolopesii was tested in media containing various sugars as carbon and energy sources. Of the sugars tested, only D-glucose, D-fructose, and D-mannose supported growth of the yeast. In media containing those sugars, the organism grew over the entire pH range tested.     

Energetics of hydrogen bonding in proteins: a model compound study.

Differences in the energetics of amide-amide and amide-hydroxyl hydrogen bonds in proteins have been explored from the effect of hydroxyl groups on the structure and dissolution energetics of a series of crystalline cyclic dipeptides. The calorimetrically determined energetics are interpreted in light of the crystal structures of the studied compounds. Our results indicate that the amide-amide and amide-hydroxyl hydrogen bonds both provide considerable enthalpic stability, but that the amide-amide hydrogen bond is about twice that of the amide-hydroxyl. Additionally, the interaction of the hydroxyl group with water is seen most readily in its contributions to entropy and heat capacity changes. Surprisingly, the hydroxyl group shows weakly hydrophobic behavior in terms of these contributions. These results can be used to understand the effects of mutations on the stability of globular proteins.     

Role of hydrogen bonding in red cell aggregation.

The role of hydrogen bonding in red cell aggregation induced by dextran was studied with the use of urea, an inhibitor for hydrogen bonding. In order to avoid hemolysis of red cells by the high concentration of urea, the studies were performed on human red cells hardened in glutaraldehyde. The degree of red cell aggregation at Hct = 45% was estimated by the use of a coaxial cylinder viscometer. The viscometric aggregation index (VAI) was calculated from viscosity values at shear rates of 52 sec-1 (eta H) and 0.05 sec-1 (eta L); VAI = (eta L - eta H)/eta H. Red cells with surface charge intact and with charge removal by neuraminidase treatment were studied. Urea at high concentrations, e.g., 6 M, significantly inhibited red cell aggregation induced by dextran. These findings indicate that hydrogen bonding plays an important role in dextran-induced red cell aggregation. An understanding of the nature of the forces involved in red cell aggregation serves to establish the physicochemical principles of cell-to-cell interactions induced by macromolecules.     

Carbon and hydrogen metabolism of xylitol and various sugars in human erythrocytes.

1) Erythrocytes are able to metabolize D-ribose, D-xylitol, D-xylulose, D-fructose and D-glucose; the rates of metabolism increase in that order from 2430 to 26200 ng atom C/ml packed cells per 120 min of incubation. 2) The utilization of the carbon of these substrates and its recovery in the products were found to be in balance, when the change in the 2,3-bisphosphoglycerate concentration was taken into account. 3) The metabolic rates strongly affected the 2,3-bisphosphoglycerate level. Without addition of substrate the decomposition rate of this intermediate was found to be 1030 nmol/ml packed cells per 120 min. 4) The net decrease of the 2,3-bisphosphoglycerate concentration and the conversion of this compound into lactate provides a NAD regeneration system which enables the red blood cells to utilize xylitol. 5) The rate of carbon metabolism via the pentose phosphate cycle is determined by the NADPH requirement of the erythrocytes which was found to be 160 nmol/ml packed cells per 120 min under the experimental conditions employed.     

A catalytic diad involved in substrate-assisted catalysis: NMR study of hydrogen bonding and dynamics at the active site of phosphatidylinositol-specific phospholipase C

Phosphatidylinositol-specific phospholipase Cs (PI-PLCs, EC 3.1.4.10) are ubiquitous enzymes that cleave phosphatidylinositol or phosphorylated derivatives, generating second messengers in eukaryotic cells. A catalytic diad at the active site of Bacillus cereus PI-PLC composed of aspartate-274 and histidine-32 was postulated from the crystal structure to form a catalytic triad with the 2-OH group of the substrate [Heinz, D. W., et al. (1995) EMBO J. 14, 3855-3863]. This catalytic diad has been observed directly by proton NMR. The single low-field line in the 1H NMR spectrum is assigned by site-directed mutagenesis: The peak is present in the wild type but absent in the mutants H32A and D274A, and arises from the histidine Hdelta1 forming the Asp274-His32 hydrogen bond. This hydrogen is solvent-accessible, and exchanges slowly with H2O on the NMR time scale. The position of the low-field peak shifts from 16.3 to 13.8 ppm as the pH is varied from 4 to 9, reflecting a pKa of 8.0 at 6 degrees C, which is identified with the pKa of His32. The Hdelta1 signal is modulated by rapid exchange of the Hepsilon2 with the solvent. Estimates of the exchange rate as a function of pH and protection factors are derived from a line shape analysis. The NMR behavior is remarkably similar to that of the serine proteases. The postulated function of the Asp274-His32 diad is to hydrogen-bond with the 2-OH of phosphatidylinositol (PI) substrate to form a catalytic triad analogous to Asp-His-Ser of serine proteases. This is an example of substrate-assisted catalysis where the substrate provides the catalytic nucleophile of the triad. This hydrogen bond becomes shorter as the imidazole is protonated, suggesting it is stronger in the transition state, contributing further to the catalytic efficiency. The hydrogen bond fits the NMR criteria for a short, strong hydrogen bond, i.e., a highly deshielded proton resonance, bond length of 2.64 +/- 0.04 A at pH 6 measured by NMR, a D/H fractionation factor significantly lower than 1.0, and a protection factor > or = 100.     

Use of uncoupling acridine dyes as stoichiometric energy probes in chloroplasts

In a suspension of spinach chloroplasts the fluorescence of atebrin and other uncoupling acridine dyes is quenched upon energization which is associated with a proportional binding of the dyes to the organelles. There is a stoichiometric relation between the amount of dye bound and the actual steady state level of energy. When the concentration of atebrin is increased in energized chloroplasts the fluorescence is completely quenched until a certain concentration is attained above which the response sharply declines. Such titrations with atebrin were carried out under conditions of partial electron transport governed by photosystems I and II, in the presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and cyanide, respectively, and of complete electron transport governed by the two photo-systems. The sum of the saturating amounts of atebrin obtained in these partial electron flow systems equals that obtained in the complete system. This lends strong support to the view that two sites of energy conservation are coupled to the linear photosynthetic electron transport.     

Modest catalysis of the decarboxylation of orotate by hydrogen bonding: a theoretical model for orotidine- 5' -monophosphate decarboxylase

As a model for interactions present in the active site of orotidine-5'-monophosphate decarboxylase (ODCase), the effect of hydrogen bonds to the carbonyl groups (O-2 and O-4) of orotic acid and its decarboxylation product was probed with ab initio calculations. We have found that the transition state/carbanion intermediate is a better proton receptor and therefore, the hydrogen bonds can be a modest source of catalysis. Comparison of the calculated data with results from site-directed mutagenesis provides some insights into the polarity of the active site.     

Binding of cations in Bacillus subtilis phosphoribosyldiphosphate synthetase and their role in catalysis.

The binding sites for the two cations essential for the catalytic function of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthases have been identified from the structure of the Bacillus subtilis phosphoribosyldiphosphate synthetase (PRPPsase) with bound Cd(2+). The structure determined from X-ray diffraction data to 2.8-A resolution reveals the same hexameric arrangement of the subunits that was observed in the complexes of the enzyme with the activator sulfate and the allosteric inhibitor ADP. Two cation binding sites were localized in each of the two domains of the subunits that compose the hexamer; each domain of the subunit has an associated cation. In addition to the bound Cd(2+), the Cd(2+)-PRPPsase structure contains a sulfate ion in the regulatory site, a sulfate ion at the ribose-5-phosphate binding site, and an AMP moiety at the ATP binding site. Comparison of the Cd(2+)-PRPPsase to the structures of the PRPPsase complexed with sulfate and mADP reveals the structural rearrangement induced by the binding of the free cation, which is essential for the initiation of the reaction. The comparison to the cPRPP complex of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli, a type I phosphoribosyltransferase, provided information about the binding of PRPP. This strongly indicates that the binding of both substrates must lead to a stabilized conformation of the loop region, which remains unresolved in the known PRPPsase complex structures.     

Stimulated and co-operative electron transfer in energy conversion and catalysis.

A new mechanism of electron transfer, stimulated electron transfer, is postulated, in which an electronic feedback is drastically increasing both the rate of electron transfer and the propagation of free energy along electron transferring molecular pathways. In principle, the idea of pushing a system far from equilibrium to achieve a high reaction rate and co-operative phenomena is applied to molecular electron transfer. The effect is calculated from a semiclassical kinetic model of a chain redox reaction with autocatalytic feedback on individual rate constants, where the steps have subsequently been minimized to obtain a continuous electron transfer pathway with electronic feedback. The influence of inhomogeneities and asymmetries in the electron transfer path and of vectorial components (electrical field, gradient of redox potential) are discussed as well as the acceleration of individual and multiple electron transfer as a function of feedback. Examples of autocatalytic feedback are provided including mechanisms involving electron transfer proteins and multi-centre electron transfer catalysts. Such a phenomenon can be described for molecular and interfacial electron transfer in analogy to stimulated and coherent light emission. The results suggest that autocatalytic or stimulated electron transfer may be a key to the understanding of efficient electron transfer and co-operative multi-electron transfer catalysis in biology and a challenge for fuel production mechanisms in artificial photosynthesis and fuel cycles.     

Hydrogen bonding of flavoprotein. I. Effect of hydrogen bonding on electronic spectra of flavoprotein.

The effect of hydrogen bonding on the transition energy and the oscillator strength of the isoalloxazine nucleus of flavins was studied by the molecular orbital method. Among the possible hydrogen bondings examined, characteristic spectral shifts were found for the hydrogen bondings at N(1) and N(5) of the nucleus. The hydrogen bonding at N(1) resulted in the shift of the first absorption band towards blue and that of the second one towards red. On the other hand, the hydrogen bonding at N(5) resulted in the shifts of both the first and the second band towards red. The spectral characteristics reported on Clostridium MP and Desulfovibrio vulgaris flavodoxin coincided with the calculated results. The application of the calculated results to D-amino acid oxidase (D-amino acid: oxygen oxidoreductase (deaminating), EC 1.4.3.3) led to the conclusion that hydrogen bonding occurs at O(12), N(3)H, O(14) and N(5) of the isoalloxazine nucleus. The occurrence of hydrogen bondings at O(12), N(3)H, and O(14) is favorable for N(5) of the isoalloxazine nucleus to accept electron from an electron donor.     

Thioamide substrate probes of metal-substrate interactions in carboxypeptidase A catalysis

Three thioamide peptides in which the oxygen atom of the scissile peptide bond is replaced by sulfur (denoted by (= S)) were synthesized and found to be good, convenient substrates for carboxypeptidase A. The thioamide bond absorbs strongly in the ultraviolet region, and enzymatic hydrolysis is monitored easily using a continuously recording spectrophotometric assay. The reaction follows Michaelis-Menten kinetics with kcat values of 68, 9.0, and 3.7 sec-1 and Km values of 0.83, 0.81, and 0.53 mM for Z-Glu-Phe(= S)-Phe, Z-Gly-Ala(= S)-Phe, and Z-Phe(= S)-Phe, respectively. Activities of the thioamides and their oxygen amide analogs were determined with a series of metal-substituted carboxypeptidases. The Cd(II), Mn(II), Co(II), and Ni(II) enzymes exhibit 30%-35%, 60%-85%, 150%-190%, and 40%-55% of the Zn(II) enzyme activity with the amide substrates; this compares with 240%-970%, 0%-15%, 340%-840%, and 30%-140% of the Zn(II) activity, respectively, with the thioamides. The activity of the Cu(II) and Hg(II) enzymes is less than 3% toward all substrates. Cadmium, a thiophilic metal, yields an enzyme which is exceedingly active with the thioamides; the kcat/Km values are 2.4-9.7-fold higher than with Zn(II) carboxypeptidase. In contrast, Mn(II), which has a relatively low affinity for sulfur, yields an enzyme with correspondingly low activity toward the thioamides. The results are consistent with a mechanism for peptide bond hydrolysis in which the metal atom interacts with the substrate carbonyl atom during catalysis.