Calorimetric studies of oxyhemoglobin dissociation. I. Reaction of sodium dithionite with oxygen

Calorimetric studies of the reduction of free oxygen in solution by sodium dithionite are in agreement with a stoichiometry of 2 moles Na₂S₂O₄ per mole of oxygen. The reaction is biphasic with ΔH_t - 118±7 kcal mol^?1 (?494 ± 29 kJ mol^?1). The initial phase of the reaction proceeds with an enthalpy change of ca ?20 kcal (?84 kJ) and occurs when 0.5 moles of dithionite have been added per mole dioxygen present. This could be interpreted as the enthalpy change for the addition of a single electron to form the superoxide anion. Further reduction of the oxygen to water by one or more additional steps is accompanied by an enthalpy change of ca ?100 kcal (?418. 5 kJ). Neither of these reductive phases is consistent with the formation of hydrogen peroxide as an intermediate. The reduction of hydrogen peroxide by dithionite in 0.1 M phosphate buffer, pH 7.15, is a much slower process and with an enthalpy change of ca ? 74 kcal mol^?1 (?314 kJ mol^?1). Dissociation of oxyhemoglobin induced by the reduction of free oxygen tension with dithionite also shows a stoichiometry of 2 moles dithionite per mole oxygen present and an enthalpy change of ca. ?101 ±9 kcal mol^?1 (?423± 38 kJ mol^?1). The difference in the observed enthalpies (reduction of dioxygen vs. oxyhemoglobin) has been attributed to the dissociation of oxyhemoglobin, which is 17 kcal mol^?1 (71 kJ mol^?1).     

Thermodynamics and kinetics of the helix-coil transition of oligomers containing GC base pairs

Absorbance-temperature profiles have been determined for the following self-complementary oligonucleotides or equimolar paris of complementary oligonucleotides containing GC base pairs: A₂GCU₂, A₃GCU₃, A₄GCU₄, A₆CG + CGU₆, A₈CG + CGU₈, A₄G₂ + C₂U₄, A₅G₂ + C₂U₅, A₄G₃ + C₃U₄, and A₅G₃ + C₃U₅. In all cases cooperative melting transitions indicate double-helix formation. As was found previously, the stability of GC containing oligomer helices is much higher than that of AU helices of corresponding length. Moreover, helices with the same length and base composition but different sequences also have quite different stabilites. The melting curves were andlyzed using a zipper model and the thermodynamic parameters for the AU pairs determined previously. The effect of single-strand stacking was considered separately. According to this model, the formation of a GC pair from unstacked single strands is associated with an ethalpy change of ?15 kcal/mole. Due to the high degree of single-strand stacking at room temperature the enthalpy change for the formation of GC pairs from unstacked single strands is only ?5 to ?6 kcal/mole. (The corresponding parameters for AU pairs are ?10.7 kcal/mole and ?5 to ?6 kcal/mole.) The sequence dependence of helix stability seems to be primarily entropic since no differences in ΔH were seen among the sequence isomers. The kinetics of helix formation was investigated for the same molecules using the temperature jump technique. Recombination of strands is second order with rate constants in the range of 10⁵ to 10⁷M^?1 sec^?1 depending on the chain length and the nucleotide sequence. Within a series of oligomers of a given type, the rates of recombination decrease with increasing chain length. Oligomers with the sequence A_nGCU_n recombine six to eight times slower than the other oligomers of corresponding chain length. The experimental enthalpies of activation of 6 to 9 kcal/mole suggest a nucleation length of one or two GC base pairs. The helix dissociation process has rate constants between 0.5 and 500 sec^?1 and enthalpies of activation of 25 to 50 kcal/mole. An increase of chain length within a given nucleotide series leads to decreased rates of dissociation and increased enthalpies of activation. An investigation of the effect of ionic strength on A_nGCU_n helix formation showed that the rates of recombination increase considerably with increased ionic strength.     

Thermodynamic and kinetic parameters of an oligonucleotide hairpin helix

The thermodynamics of the hairpin helix-single strand transition of A₆C₆U₆ has been analyzed by a staggering zipper model with consideration of single strand stacking. This analysis yields an enthalpy change of +11 kcal/mole for the formation of a first, isolated base pair. The stability constant of a first (intramolecular) base pair in A₆C₆U₆ is around 2 × 1O^?5 at 25°C, whereas a first (intermoleciilar) base pair in an A₆ · U₆ helix is characterised by a stability constant of about 4 × 10^?3M^?1 (25°C, extrapolated from A_n · V_n oligomer measurements). These data indicate a destabilizing effect of the C₆ loop.The rate constant of hairpin helix formation is 2 to 3 × 10⁴ sec^?1 associated with an activation enthalpy of +2.5 kcal/mote. The rate of helix dissociation of the A₆C₆U₆ hairpin is in the range of 10³ to lO⁵ sec^?1 with an activation enthalpy of 21 $\text{kcal}\text{mole}$. A comparison with the kinetic parameters obtained for A · U oligomer helices shows a specific influence of the C₆ loop due to the stacking tendency of the cytosine residues. This intluence is preferentially reflected in the relatively low value of the rate constant of helix formation.     

The enthalpy of oxidation of flavin mononucleotide

The oxidation enthalpy of reduced flavin mononucleotide at pH 7.0 in 0.2 m phosphate buffer has been studied by determining the heat associated with the reaction: FMNH₂ + 2 Fe(CN)^?3₆ ? FMN + 2 Fe(CN)^?4₆ + 2 H⁺. (a) (The quinone, semiquinone, and hydroquinone forms of FMN are represented as FMN, FMNH, and FMNH₂, respectively.) Calorimetric experiments were performed in a flow microcalorimeter which was modified to prevent sample contamination by oxygen. The enthalpy observed for reaction (a), after correction for dilution and buffer effects, was ?39.2 ± 0.4 kcal (mole FMNH₂)^?1 at 25 °C. The potential difference, ΔE′, developed by reaction (a) was determined potentiometrically and corresponded to a free energy change, ΔG′, of ?30.3 kcal (mole FMNH₂)^?1. The resulting entropy change, ΔS′, was thus calculated to be ?29.8 e.u. Reaction (a) was also studied at temperatures of 7 °C and 35.5 °C. ΔC_p′ for the reaction was calculated as ?155 ± 18 cal deg^?1 (mole FMNH₂)^?1 at 20 °C. ΔH′ for the reaction (b), FMNH₂ ? FMN + H₂, (b) was calculated as +14.2 ± 0.7 kcal mole^?1 at 25 °C, relative to the enthalpy of the hydrogen electrode being identically equal to zero at all values of pH and temperature. The free energy at pH 7.0 for reaction (b), calculated from the potential was found to be ?9.7 kcal mole^?1, which resulted in an entropy for reaction (b) of 80.2 e.u. A thermal titration of reaction (a) was used to calculate the thermodynamic parameters for the formation of semiquinone dimer according to the reaction FMNH₂ + FMN ? (·FMNH)₂. (c) The free energy, enthalpy, and entropy changes for reaction (c) were estimated to be ?6.1 kcal mole^?1, ?7 kcal mole^?1, and ?3 e.u., respectively.     

Recognition of a Flipped Base in a Hairpinloop DNA by a Small Peptide

Two tiny hairpin DNAs, CORE (dAGGCTTCGGCCT) and AP2 (dAGGCTXCGGCCT; X: abasic nucleotide), fold into almost the same tetraloop hairpin structure with one exception, that is, the sixth thymine (T6) of CORE is exposed to the solvent water (Kawakami, J. et al., Chem. Lett. 2001, 258–259). In the present study, we selected small peptides that bind to CORE or AP2 from a combinatorial pentapeptide library with 2.5 × 10⁶ variants. On the basis of the structural information, the selected peptide sequences should indicate the essential qualifications for recognition of the hairpin loop DNA with and without a flipped base. In the selected DNA binding peptides, aromatic amino acids such as histidine for CORE and glutamine/aspartic acid for AP2 were found to be abundant amino acids. This amino acid preference suggests that CORE-binding peptides use π–π stacking to recognize the target while hydrogen bonding is dominant for AP2-binding peptides. To investigate the binding properties of the selected peptide to the target, surface plasmon resonance was used. The binding constant of the interaction between CORE and a CORE-binding peptide (HWHHE) was about 1.1 × 10⁶ M^?1 at 25°C and the resulting binding free energy change at 25°C (ΔG°₂₅) was ?8.2 kcal mol^?1. The binding of the peptide to AP2 was also analyzed and the resulting binding constant and ΔG°₂₅ were about 4.2 × 10⁴ M^?1 and ?6.3 kcal mol^?1, respectively. The difference in the binding free energy changes (ΔΔG°₂₅) of 1.9 kcal mol^?1 was comparable to the values reported in other systems and was considered a consequence of the loss of π–π stacking. Moreover, the stabilization effect by stacking affected the dissociation step as well as the association step. Our results suggest that the existence of an aromatic ring (T6 base) produces new dominant interactions between peptides and nucleic acids, although hydrogen bonding is the preferable mode of interaction in the absence of the flipping base. These findings regarding CORE and AP2 recognition are expected to give useful information in the design of novel artificial DNA binding peptides.     

Interacalation of ethidium ion into DNA and RNA oligonucleotides

The thermodynamics of ethidium ion binding to the double strands formed by the ribooligonucleotides rCA₅G + rCU₅G and the analogous deoxyribo-oligonucleotides dCA₅G + dCT₅G were determined by monitoring the absorbance versus temperature at 260 and 283 nm at several concentrations of oligonucleotides and ethidium bromide. A maximum of three ethidium ions bind to the oligonucleotides, which is consistent with intercalation and nearest-neighbor exclusion. For the ribo-oligonucleotide the binding mechanism is complex. Either two sites (assumed to be the intercalation sites at the two ends of the oligonucleotide) bind more strongly by a factor of 140 than the third site, or all sites are identical, but there is strong anticooperativity on binding (cooperativity parameter, 0.1). In sharp contrast, the binding to the same sequence (with thymine substituted for uracil) in the deoxyribo-oligonucleotide showed all sites equivalent and no cooperativity. For the ribo-oligonucleotides the enthalpy for ethidium binding is ?14 kcal/mol. The equilibrium constants at 25°C depend on the model; either K = 6 × 10⁵M^?1 for the two strong sites (4 × 10³M^?1 for the weak site) or K = 2.5 × 10⁵M^?1 for the intrinsic constant of the anticooperative model. For the equivalent deoxyribo-oligonucleotide the enthalpy of binding is -9 kcal/mol and the equilibrium constant at 25°C is a factor of 10 smaller (K = 2.5 × 10⁴M^?1).     

The effect of monovalent cations on the association behavior of guanosine 5'-monophosphate, cytidine 5'-monophosphate, and their equimolar mixture in aqueous solution

¹H- and ³¹P-nmr spectroscopy have been used to investigate the self-association of M₂(5′-CMP) [M = Li⁺, Na⁺, K⁺, Rb⁺, or (CH₃)₄ N⁺; 5′-CMP = cytidine 5′-monophosphate], the self-association of Li₂(5′-GMP) (5′-GMP = guanosine 5′-monophosphate), and the heteroassociation of 5′-GMP and 5′-CMP (1 : 1 mole ratio) in aqueous solution as a function of the nature of the monovalent cation. Proton spectral differences for the different 5′-CMP salts exhibit a cation-size dependence and have been ascribed to a change in the stacking geometry. An average stacking association constant of 0.63 ± 0.24M^?1 at 1°C, consistent with the weak stacking interactions of the cytosine bases, was determined for the 5′-CMP salts. Heteroassociation of 5′-GMP and 5′-CMP follows the reverse of the cation order for the formation of ordered aggregates of 5′-GMP. Heteroassociation occurs in the presence of Li⁺, Na⁺, and Rb⁺ ions, but only self-association occurs for the K⁺ nucleotides. Li₂(5′-GMP), which does not form ordered species, self-associates to form disordered base stacks with a stacking constant of 1.63 ± 0.11M^?1 at 1°C.     

Kinetics of the renaturation of yeast tRNA3 leu.

Yeast tRNA₃^Leu is one of several tRNA molecules which can adopt a stable, biologically inactive, denatured conformation. The circular dichroism of the native and denatured conformers differs, providing the basis for the present study of the mechanism for the renaturation process. Conversion of the denatured structure to the native takes place in two steps: a rapid change occurring immediately on addition of Mg⁺⁺, followed by a slower, strongly temperature-dependent step which returns the molecule to its biologically active state. Optimal kinetic data for the second step could be obtained at 285 nm. Analysis of the time dependence of Δε₂₈₅ by the Guggenheim method demonstrated that this step follows first-order kinetics. The temperature dependence of the rate constants over the range 32–41°C yielded the following parameters for the rate-limiting step: E_a = 69 kcal/mole, ΔH^? = 69 kcal/mole, and ΔS^? = 146 cal/mole deg. Values of this magnitude are typical of order—order transitions in nucleic acids.     

A calorimetric investigation of the heats of binding of Mg ++ to poly A, to poly U, and to their complexes

The heats of binding of Mg⁺⁺ ions to poly A, poly U, and to their complexes, in the presence of Na⁺ ions, have been measurd calorimetrically. In all cases the heat, ΔH(θ), exhibitis a distinct dependence on the extent of binding, θ, and in the cases of poly A and poly U also on the Na⁺ concentration. The values of ΔH(θ) range from +2 to +3 kcal/mole of Mg⁺⁺ bound at θ = 0 to 1.3 kcal/mole at θ = 0.5 except in poly A where at θ = 0 ΔH(θ) = ?2 to ?3 kcal/mole. This is interpreted as being due to a facilitation of base stacking by the binding of Mg⁺⁺. The extent of facilitation is consistent with current estimates of base stacking. A similar effect but of much smaller magnitude is believed to obtain in poly A poly U. An interpretation of the dependence of ΔH(θ) on θ in terms of simple electrostatic interactions, but neglecting solvent effects, was attempted and found to be inadequate.     

Enzymatic studies on the interaction of myosin and heavy meromyosin with 1,N 6 -ethenoadenosine triphosphate ( ATP), a fluorescent analog of ATP

The fluorescent analog of adenosine triphosphate (ATP)¹ 1,N⁶-ethenoadenosine triphosphate, (εATP), has been utilized as a substitute for ATP in the myosin and heavy meromyosin ATPase systems. For myosin, the analog εATP replaced ATP with a somewhat larger K_m (2.6 × 10^?4 mole ?^?1 for εATP as opposed to 8.8 × 10^?5 mole ?^?1 for ATP), indicating that the apparent affinity of the enzyme for εATP is less than for ATP. Perhaps of more interest, further comparison yielded a V_max for εATP about two and one half times the value for ATP (20 μmole PO₄ sec^?1 g protein^?1 as opposed to 8.1 μmole sec^?1 g protein^?1). Results for the HMM-εATPase system were similar, yielding a K_m value of 1.47 × 10^?4 mole ?^?1 and a V_max of 54.2 μmole PO₄ sec^?1 g protein^?1, as opposed to corresponding K_m and V_max values of 1.23 × 10^?4 mole ?^?1 and 20.4 μmole PO₄ sec^?1 g protein^?1, respectively for the HMM-ATP interaction. The pH dependence of εATPase for both systems was comparable to ATP, suggesting a similarity in the mechanism of hydrolysis of the two nucleotides. Activation of εATPase by Ca²⁺ in the presence of 0.5 M KCl was comparable to ATPase for both systems, but inhibition by Mg²⁺ seemed to be more effective for εATPase. These results indicate that εATP is an excellent substitute for ATP in the myosin and heavy meromyosin systems and because of its insertion into the active site of these muscle proteins, it promises to be a very useful probe for conformation studies at this level.     

Analysis of the binding of phenylalanine to phenylalanyl-tRNA synthetase

Using the complete rate equation for the PP_i-ATP exchange reaction at equilibrium, the dissociation constants of phenylalanine (10^?5m), phenylalanine butyl ester (8 × 10^?5m), benzyl alcohol (6 × 10^?4m), phenylalaninol (2 × 10^?4m), hydrocinnamic acid (3 × 10^?3m) and glycine (>1 m) with the phenylalanyl-tRNA synthetase (Escherichia coli K12) were determined. Taking the model of Koshland (1962) for the estimation of the configurational free energy change due to proximity and orientation, and decomposing the process of binding into several thermodynamic steps, the contribution to binding of the benzyl group, glycine unit, protonated amino group, carboxylate group and joint interactions were estimated. The results are: (1) the standard free energy contributions for binding phenylalanine are benzyl group (?8.2 kcal/mol), glycine unit (?2.5 kcal/mol), protonated amino group (?0.8 kcal/mol) and carboxylate group (1 kcal/mol). (2) The standard free energy change due to the change in the interaction between the protonated amino group and carboxylate group when they are transferred from the aqueous environment to the enzyme environment is ?2.7 kcal/mol. (3) A dissociation constant for glycine of 7.5 m is calculated without the hypothesis that a conformational change occurs in the enzyme when the benzyl unit of phenylalanine binds, permitting an interaction of the enzyme with the protonated amino and/or carboxylate groups.The detection of E·AA² and E·ATP shows that a sequential addition of substrates is not necessary for binding. A comparison of the dissociation constants of E·AA (10^?5m), E·ATP (1.5 × 10^?3m), E·PP (5.5 × 10^?4m), E·I (8 × 10^?5m) and the mixed complexes E·I·ATP (6 × 10^?8m²), E·I·PP (5 × 10^?8m²) and E·AA·PP (7 × 10^?9m²), with phenylalanine butyl ester as the inhibitor, indicates no strong interaction between the binding of ATP or PP_i with the binding of phenylalanine.     

A facile synthesis of ceramides.

Lateral diffusion of phosphatide molecules in liquid crystalline bilayers has been analysed as a case of co-operative lattice diffusion. The potential energy of interaction between two molecules is assumed to arise from Van der Waals interactions of the hydrocarbon chains, and to have the form suggested by Salem [6]. From the observed values of the self-diffusion constant (of the order of 10^?8 cm² sec^?1) the depth of the potential “well” for two molecules at the equilibrium separation was estimated to have a lower limit of 1.95 kcal per mole, and the energy barrier to lateral motion was estimated to have an upper limit of 7.21 kcal per mole.     

Cooperative nonenzymic base recognition II. thermodynamics of the helix-coil transition of oligoadenylic + oligouridylic acids

The properties of oligonucleotide helices of adeuylic- and uridylic acid oligomers have been investigated by measurements of hypo-and hyperchromieity. High ionic strengths favor the formation of triple helices. Thus, the double helix-coil transition can be studied (without interference by triple helices) only at low ionic-strength. A “phase diagram” is given representing the T_m-values of the various transitions at different ionic strengths for the system A(pA)₁₇ + U(pU)₁₇. Oligonucleolides of chain lengths <8 always form both double and triple helices at the nucleotide concentrations required for base pairing. For this reason the double helix-coil transition without coupling of the triple helix equilibrium can only be measured for chain lengths higher than 7. Melting curves corresponding to this transition have been determined for chain lengths 8, 9, 10, 11, 14 and 18 at different concentrations. An increase in nucleotide concentration leads to an increase in melting temperature. The shorter the chain length the lower the T_m-value and the broader the helix-coil transition. The experimental transition curves have been analysed according to a staggering zipper model with consideration of the stacking of the adeuylic acid single strands and the electrostatic repulsion of tlip phosphate charges on opposite strands. The temperature dependence of the nucleation parameter has been accounted for by a slacking factor x. The stacking factor expresses the magnitude of the stacking enthalpy. By curve fitting xwas computed to be 0.7, corresponding to a stacking enthalpy of about S kcal/mole. The model described allows the reproduction of the experimental transition curves with relatively high accuracy. In an appendix the thermodynamic parameters of the stacking equilibrium of poly A and of the helix-coil equilibria of poly A + poly U at neutral pH are calculated (ΔH_A = ?7.9 kcal/mole for the poly A stacking and ΔH₁₂ = ?10.9 kcal/mole for the formation of the double helix from the randomly coiled single strands). A formula for the configurational entropy of polymers derived by Flory on the basis of a liquid lattice model is adapted to calculate the stacking entropies of adenylic oligomers.     

The Influence of Nearest Neighbours on the Efficiency of Coaxial Stacking at Contiguous Stacking Hybridization of Oligodeoxyribonucleotides

Contiguous stacking hybridization of oligodeoxyribonucleotides with a stem of preformed minihairpin structure of a DNA template was studied with the use of UV‐melting technique. It was shown that the free‐energy of the coaxial stacking interaction (ΔG°_ST at 37°C, 1 M NaCl, pH 7.4) at the complementary interface XA^*pTY/ZATV (an asterisk stands for a nick) strongly depends on the type of nearest neighbor bases X and Y flanking the nicked dinucleotide step. The maximum efficiency of the coaxial stacking was observed for the PuA^*pTPy/PuATPy interface, whereas the minimum efficiency was obtained for the PyA^*pTPu/PyATPu interface. A 5′‐phosphate residue in the nick enhances the coaxial stacking. In dependence on duplex structure the observed efficiency of A^*T/AT coaxial stacking varied from (? 0.97 kcal/mol) for unphosphorylated TA^*TA/TATA interface to three‐fold higher value (? 2.78 kcal/mol) for GA^*pTT/AATC interface.     

pH-Dependent Raman spectra and thermal melting profiles for polycytidylic acid

The Raman spectrum of polycytidylic acid was investigated in the pH range of 6.6–4.1. The thermal melting temperatures and the nature of the thermal melting profiles change in this range as monitored by the three Raman band envelopes, which include the 780-, 805-cm^?1 bands, the 1190-, 1285-cm^?1 bands, and the 1527-cm^?1 band. By coupling these data with the theory of Raman scattering intensity and quantitative pH profiles for cytidine, it is shown that the band envelopes studied exhibit specific, yet different information regarding the thermal melting process. The band envelopes at 1170–1310 and 1527 cm^?1, which are a sensitive function of both the extent of protonation and base stacking (hypochromic), reveal T_m values which agree with values derived from uv melting profiles. The 760–830-cm^?1 envelope, which is not directly sensitive to cytosine residue protonation, but includes information associated with base stacking (the 780-cm^?1 band) and the nature of the phosphodiester backbone (the frequency-dependent 805-cm^?1 component), exhibits T_m values which deviate from the values obtained from the other bands. The observed differences are pH-dependent and correlate well with the extent of deprotonation that takes place in the denaturation process. Details of the spectrum of neutral and protonated poly(C) from pH 7 to 4.1 are discussed and related to the nature of the thermal denaturation process.     

Titles of related papers in other sections

Arrhenius plots of rabbit skeletal muscle sarcolemmal Na⁺,K⁺-ATPase contain no temperature breaks. The apparent activation energy (22.8 kcal/mole in the presence of 1 mM MgCl₂ or 15.9 kcal/mole in the presence of 3 mM MgCl₂) does not depend on the Na⁺/K⁺ ratio in the incubation medium, but decreases in the presence of anserine (instead of Tris buffer).     

Relaxation kinetics of the helix-coil transition of a self-complementary ribo-oligonucleotide: A7U7

Relaxation measurements on the kinetics of the double helix to coil transition for the self-complementary ribo-oligonucleotide A₇U₇ are reported over a concentration range of 6.9 μM to 19.6 μM in single strand in 1 M NaCl. The rate constants for helix formation are about 2 × 10⁶ M^?1 s^?1 and decrease with increasing temperature yielding an activation enthalpy of ?6 $\text{kcal}\text{mole}$. The rate constants for helix dissociation range from 3 to 250 s^?1 and increase with increasing temperature yielding an activation enthalpy of +45 $\text{kcal}\text{mole}$. The kinetic data reported here for 1 M NaCl is compared with previously published results obtained at lower salt concentrations. These data are discussed in terms of the quantitative effect of ionic strength on the kinetics of helix-coil transitions in oligo- and polynucleotides.     

Ordered substrate binding and evidence for a thermally induced change in mechanism for E. coli aspartate transcarbamylase

Isotopic exchange kinetics at equilibrium for E. coli native aspartate transcarbamylase at pH 7.8, 30 °C, are consistent with an ordered BiBi substrate binding mechanism. Carbamyl phosphate binds before l-Asp, and carbamyl-aspartate is released before inorganic phosphate. The rate of [¹⁴C]Asp C-Asp exchange is much faster than [³²P]carbamyl phosphate P_i exchange. Phosphate, and perhaps carbamyl phosphate, appears to bind at a separate modifier site and prevent dissociation of active-site bound P_i or carbamyl phosphate. Initial velocity studies in the range of 0–40 °C reveal a biphasic Arrhenius plot for native enzyme: E_a (>15 °C) = 6.3 kcal/ mole and E_a (<15 °C) = 22.1 kcal/mole. Catalytic subunits show a monophasic plot with E_a ? 20.2 kcal/mole. This, with other data, suggests that with native enzyme a conformational change accompanying aspartate association contributes significantly to rate limitation at t > 15 °C, but that catalytic steps become definitively slower below 15 °C. Model kinetics are derived to show that this change in mechanism at low temperature can force an ordered substrate binding system to produce exchange-rate patterns consistent with a random binding system with all exchange rates equal. The nonlinear Arrhenius plot also has important consequences for current theories of catalytic and regulatory mechanisms for this enzyme.     

The binding of adenosine to polyuridylic acid in which uracil residues are chemically altered.

The binding of adenosine-¹⁴C to polyuridylic acid (poly(U)) and several modified poly(U)s has been studied by equilibrium dialysis. The poly(U) was modified by addition of appropriate reagents across the 5,6-double bond of the uracil ring to form the photohydrate, photodimer, dihydrouracil, the HOBr addition product and the HSO₃^? addition product. Modification of the uracil rings decreases the amount of adenosine which can be bound to the poly(U); the decrease in binding is a function of the fraction of uracil rings which have been changed. Using the expression S = S₀(1 ? αr)² to relate the fraction of uracil rings modified (r) to the number of binding “sites” remaining (S), it is found that α is about 1 for all the modifications except photodimer where it is about 2. These observations are taken to mean that the loss of binding capacity of the poly(U) resulting from modifications of the uracil ring is caused by loss of planarity of the uracil rings caused by the modifications, and consequent loss of double helix structure, but that for all modifications except photodimer there is no disruption of the poly(U) double helix on either side of the leison. There does appear to be local melting on either side of the photodimer lesion. The sigmoidal binding isotherms (A_b versus C_a) of modified and unmodified poly(U) can be approximated closely by the following equation: ((1)) (1) where A_b = bound A, C_a = free A, n = minimum number of adjacent A′s in complex, S = concentration of sites on poly(U), and K₁ = (K_m)^1/m for all m ≥ n. The stacking energy of adenosine (w) can be calculated accurately using the following equation, where dθ/d ln C_a is obtained from Eq. (1). ((2)) (2) For unmodified poly(U), w is ?2.0 kcal/mole and ΔG° (?;RT ln K₁) is ?3.2 kcal/mole. The difference (?1.2 kcal/mole) is attributed to hydrogen bonding. Heavily photohydrated poly(U) does not bind guanosine or guanosine-5′-phosphate.