NMR Line-broadening Techniques Research Articles

Comparison of the Self-Exchange and Interfacial Charge-Transfer Rate Constants for Methyl- versus tert-Butyl-Substituted Os(III) Polypyridyl Complexes

Differences in the self-exchange and interfacial electron-transfer rate constants have been evaluated for a relatively unhindered Os(III/II) redox system, osmium(III/II) tris(4,4'-di-methyl-2,2'-bipyridyl), [Os(Me2bpy)3]3+/2+, relative to those of a relatively hindered system, osmium(III/II) tris(4,4'-di-tert-butyl-2,2'-bipyridyl), [Os(t-Bu2bpy)3]3+/2+. In contrast to the predicted increase in rate constant by a factor of 2-3 due to the difference in reorganization energy of the two complexes, introduction of the tert-butyl functionality decreased the self-exchange rate constant, as measured by NMR line-broadening techniques, by a factor of approximately 50 as compared to that of the analogous methyl-substituted osmium complex. Steady-state current density versus potential measurements, in conjunction with differential capacitance versus potential measurements, were used to compare the interfacial electron-transfer rate constants at n-type ZnO electrodes of [Os(t-Bu2bpy)3]3+/2+ and [Os(Me2bpy)3]3+/2+. The interfacial electron-transfer rate constant for the reduction of [Os(t-Bu2bpy)3]3+ was 100 times smaller than that for [Os(Me2bpy)3]3+. The results indicate that the tert-butyl group can act as a spacer on an outer-sphere redox couple and significantly decrease the electronic coupling of the electron-transfer reaction in both self-exchange and interfacial electron-transfer processes.

Use of Phosphorus Ligand NMR Probes To Investigate Electronic and Second-Sphere Solvent Effects in Ligand Substitution Reactions at Manganese(II) and Manganese(III)

Manganese/ligand association dynamics were studied using a series of structurally related anionic phosphorus ester ligand probes [CH(3)OP(O)(X)(Y)(-), where X = CH(3)O, CH(3)CH(2), or H and Y = O, S, or BH(3)]. Reactions of the probe ions with Mn(H(2)O)(6)(2+) and a manganese(III) porphyrin (Mn(III)TMPyP(5+)) were studied in aqueous solution by paramagnetic (31)P NMR line-broadening techniques. A satisfactory linear free energy relationship for reactions of the probe ions with Mn(H(2)O)(6)(2+) and Mn(III)TMPyP(5+) required consideration of both the basicity and solvent affinity of the probe ligands: log(k(app)) = log(k(0)) + alpha pK(a) + beta log(K(ext)), where k(0), alpha, and beta are metal complex dependent parameters and pK(a) and K(ext) represent the measured Bronsted acidity and water/n-butanol extraction constant for the probe anions, respectively. Reactions of Mn(H(2)O)(6)(2+) were relatively insensitive to changes in ligand basicity (alpha = -0.04) and favored the more hydrophilic anions (beta = -0.54). These observations are consistent with a dissociative ligand exchange mechanism wherein the outer-sphere complex is stabilized by hydrogen bonding between Mn(H(2)O)(6)(2+) and the incoming ligand. In contrast, reactions with Mn(III)TMPyP(5+) are accelerated by decreases in both the basicity (alpha = -0.43) and the hydrophilicity (beta = +0.97) of the probe. We conclude that reactions of Mn(III)TMPyP(5+) are also dissociative but that the aromatic groups of the porphyrin provide a hydrophobic environment surrounding the ligand binding site in Mn(III)TMPyP(5+). Thus, the probe/water solvent interactions must be significantly weakened in order to form the outer-sphere complex that leads to ligand substitution. This work demonstrates the utility of phosphorus relaxation enhancement (PhoRE) techniques for characterizing the second coordination sphere environment of metal complexes leading to ligation and will allow comparison of the second coordination spheres of Mn(H(2)O)(6)(2+) and Mn(III)TMPyP(5+) to those of other metal complexes.

Membrane Dynamics of the Amphiphilic Siderophore, Acinetoferrin

Acinetobacter haemolyticus is an antibiotic resistant, pathogenic bacterium responsible for an increasing number of hospital infections. Acinetoferrin (Af), the amphiphilic siderophore isolated from this organism, contains two unusual trans-2-octenoyl hydrocarbon chains reminiscent of a phospholipid structural motif. Here, we have investigated the membrane affinity of Af and its iron complex, Fe-Af, using small and large unilamellar phospholipid vesicles (SUV and LUV) as model membranes. Af shows a high membrane affinity with a partition coefficient, K(x)= 6.8 x 10(5). Membrane partitioning and trans-membrane flip-flop of Fe-Af have also been studied via fluorescence quenching of specifically labeled vesicle leaflets and (1)H NMR line-broadening techniques. Fe-Af is found to rapidly redistribute between lipid and aqueous phases with dissociation/partitioning rates of k(off) = 29 s(-1) and k(on) = 2.4 x 10(4) M(-1) s(-1), respectively. Upon binding iron, the membrane affinity of Af is reduced 30-fold to K'(x) = 2.2 x 10(4) for Fe-Af. In addition, trans-membrane flip-flop of Fe-Af occurs with a rate constant, k(p) = 1.2 x 10(-3) s(-1), with egg-PC LUV and a half-life time around 10 min with DMPC SUV. These properties are due to the phospholipid-like conformation of Af and the more extended conformation of Fe-Af that is enforced by iron binding. Remarkable similarities and differences between Af and another amphiphilic siderophore, marinobactin E, are discussed. The potential biological implications of Af and Fe-Af are also addressed. Our approaches using inner- and outer-leaflet-labeled fluorescent vesicles and (1)H NMR line-broadening techniques to discern Af-mediated membrane partitioning and trans-membrane diffusion are amenable to similar studies for other paramagnetic amphiphiles.

Proton exchange kinetics from the bound waters on the oxo-centered rhodium(iii) trimer [Rh3(µ3-O)(µ-O2CCH3)6(OH2)3]+: a variable pH and temperature 1H NMR study

Proton exchange from the bound to the bulk waters on the oxo-centered rhodium(III) trimer, [Rh(3)(micro(3)-O)(micro-O(2)CCH(3))(6)(OH(2))(3)](+)(abbreviated as Rh(3)(+)), was investigated over the temperature range of 219.1-313.9 K using a (1)H NMR line-broadening technique. By solving the modified Bloch equations for a two-site chemical exchange, lifetimes (tau) for proton transfer at pH = 2.7, 3.6, and 7.0 ([Rh(3)(+)]= 26 mM, T= 298 K) were determined to be 0.3 (+/-.08) ms, 2 (+/-0.3) ms, and 0.2 (+/-0.2) ms, respectively. From the temperature dependence of the rate, the activation parameters were determined to be DeltaH(++)= 16.2 (+/-0.5) kJ mol(-1) and DeltaS(++)=- 123 (+/-2) J mol(-1) K(-1), DeltaH(++)= 14.9 (+/-0.5) kJ mol(-1) and DeltaS(++)=- 141 (+/-2) J mol(-1) K(-1), and DeltaH(++)= 45 (+/-2) kJ mol(-1) and DeltaS(++)=- 22 (+/-5) J mol(-1) K(-1) for pH = 2.7, 3.6 and 7.0, respectively. All results are reported for a mixed solvent system [acetone : 250 mM NaClO(4)(aq)(3:1)], which was necessary to depress the freezing point of the solution so that the (1)H NMR signal due to bound water could be observed. The pK(a) of Rh(3)(+) was measured to be 8.9 (+/-0.2) in the mixed solvent, which is near the pK(a) for an aqueous solution (8.3 (+/-0.2)). Surprisingly, the lifetimes for protons on Rh(3)(+) are close to those observed for the Rh(OH(2))(6)(3+) ion, in spite of the considerable difference in structure, Brønsted acidity of the bound waters and average charge on the metal ion.

Kinetics and Mechanism of Formation of Yttrium Alkyl Complexes from (Cp*2YH)2 and Alkenes

The dissociation of the dimer (Cp* 2 YH) 2 (2) to the Cp* 2 YH monomer is an important process in reactions of 2 with alkenes. The rate of dissociation of 2 was measured by NMR line-broadening techniques (14 s - 1 at 0 °C, ΔG = 14.5 kcal mol- 1 , ΔH = 14.9(8) kcal mol - 1 , and ΔS = 3(2) eu). A full range of dissociative and associative mechanisms for reaction of alkenes with 2 was found. For the most crowded and least reactive alkenes studied, 2-butene and 2-methylpropene, reaction with 2 occurred slower than dissociation of dimer 2; kinetic studies established reversible dissociation of 2 to monomeric Cp* 2 YH followed by competitive trapping by alkene and recombination to regenerate 2. Kinetic studies of the less crowded alkene 3-methyl-1-butene are consistent with rate-limiting dissociation of dimer 2 followed by efficient trapping of the intermediate Cp* 2 YH by alkene. The least crowded terminal alkenes such as 1-hexene reacted with 2 at a rate faster than dimer dissociation; kinetic studies established a two-component rate law involving a second-order term for direct attack of alkene on the dimer and a first-order term involving rate-determining dimer dissociation followed by rapid alkene reaction with monomeric Cp* 2 YH. The reactions of terminal alkenes with 2 initially gave mixtures of single- and double-alkene-insertion products but no triple-insertion products. The initially formed n-alkyl yttrium complex reacts with terminal alkenes at a rate similar to the reaction of yttrium hydride dimer 2 with terminal alkenes. The more crowded β-alkyl yttrium double-insertion product is much less reactive toward terminal alkenes.

Intrinsic Barriers for Electron and Hydrogen Atom Transfer Reactions of Biomimetic Iron Complexes

Self-exchange reactions between high-spin iron complexes of 2,2'-bi-imidazoline (H2bim) have been investigated by the dynamic NMR line-broadening technique. Addition of the ferric complex (Fe III (H2bim)3) 3+ causes broadening of the 1 H NMR resonances of the ferrous analogue, (Fe II (H2bim)3) 2+ . This indicates electron self-exchange with ke - ) (1.7 ( 0.2) 10 4 M -1 s -1 at 298 K in MeCN-d3 (I ) 0.1 M). Similar broadening is observed when the deprotonated ferric complex (Fe III (Hbim)(H2bim)2) 2+ is added to (Fe II (H2bim)3) 2+ . Because these reactants differ by a proton and an electron, this is a net hydrogen atom exchange reaction. Kinetic and thermodynamic results preclude stepwise mechanisms of sequential proton and then electron transfer, or electron and then proton transfer. Concomitant electron and proton (H ¥ ) transfer occurs with bimolecular rate constant kH ¥ ) (5.8 ( 0.6) 10 3 M -1 s -1 . This is a factor of 3 smaller than ke - under the same conditions. The H-atom exchange reaction exhibits a primary kinetic isotope effect kNH/kND ) 2.3 ( 0.3 at 324 K, whereas no such effect is detected in the electron exchange reaction. Proton self-exchange between the two ferric complexes, (Fe III (Hbim)(H2bim)2) 2+ and (Fe III (H2bim)3) 3+ , has also been investigated and is found to be faster than both the electron and H-atom transfer reactions. From kinetic analyses and the application of simple Marcus theory, an order of intrinsic reaction barriers IH ¥ > Ie - > IH + is derived. The reorganization energies are discussed in terms of their inner-sphere and outer-sphere components.

Examination of Electron Transfer Self-Exchange Rates Using NMR Line-Broadening Techniques: An Advanced Physical Inorganic Laboratory Experiment

This article describes the determination of the decamethylferrocene(Fc*)/decamethylferrocenium (Fc*+) electron transfer self-exchange rate by NMR line-broadening techniques. NMR spectra of Fc*, Fc*+, and several mixtures yield frequency and peak-width data that are used to solve for the self-exchange rate constant. Students obtain rate constants in the range 1.6 x 107 to 2.4 x 107 M-1 s-1 (38 °C in acetone-d6), which is in good agreement with the literature value of 2.4 x 107 M-1 s-1 (25 °C). The experiment represents an alternative to the classic rate of amide bond rotation as a demonstration of the effectiveness of NMR in analyzing dynamic molecular processes.

Rhodium(III) and Iridium(III) Solvates of the Form [(η5-C5Me5)M(S)3]2+ as Models for the Reactivity of Half-Sandwich Compounds1

Solvent exchange on the half-sandwich organic solvates [(eta(5)-C(5)Me(5))M(S)(3)](2+) (M = Rh, S = MeCN (1) or Me(2)SO (3); and M = Ir, S = MeCN (2) or Me(2)SO (4)) has been investigated as a function of temperature, pressure, and concentration of free solvent by (1)H NMR line-broadening techniques in CD(3)CN and/or CD(3)NO(2). The exchange rates span several orders of magnitude, from k(ex)(298) = 8.8 x 10(-)(2) s(-)(1) for 2 to 3.6 x 10(3) s(-)(1) for 3, as a result of changes in the electronic and steric properties of the ligands. Nevertheless, the volume of activation remains consistently positive for compounds 1-4 with values ranging from +0.8 to +3.3 cm(3) mol(-)(1). In combination with the positive activation entropies obtained and the first-order rate law established for these systems, it was concluded that regardless of the nature of the ligand the solvent exchange process on 1-4 proceeds via a dissociative D mechanism. Of note, the intermolecular exchange with free Me(2)SO on 4 takes place exclusively from a conformational isomer of 4 (structure 4.2), which is itself in equilibrium with a second, more compact conformer (structure 4.1).

Formation, Properties, and Characterization of a Fully Reduced Fe(II)Fe(II) Form of Spinach (and Parsley) [2Fe-2S] Ferredoxin with the Macrocyclic Complex [Cr(15-aneN(4))(H(2)O)(2)](2+) as Reductant.

Reduction of spinach and parsley ferredoxin FdI in the Fe(III)Fe(III) state with the 1,4,8,12-tetraazacyclopentadecane complex [Cr(15-aneN(4))(H(2)O)(2)](2+), here written as Cr(II)L, provides the first evidence for two 1-equiv steps yielding an Fe(II)Fe(II) product. Rate constants (25 degrees C) for spinach FdI are 2760 and 660 M(-)(1) s(-)(1), respectively, at pH 7.5, I = 0.100 M (NaCl). An important observation is that the Cr(III)L generated in the first step remains attached to the Fe(II)Fe(III) product and perturbs the protein active site sufficiently to make the second stage possible. The second Cr(II)L reduction is of the "outer-sphere" type, and the Cr(III)L generated is not attached to the protein. Anaerobic reoxidation of the fully reduced protein with [Co(NH(3))(6)](3+) is rapid and can be achieved with approximately 80% recovery of the Fe(III)Fe(III) oxidation state over 40 min. Air oxidation yields the Cr(III)L product Fe(III)Fe(III).Cr(III)L (Fe:Cr = 2:1). With Anabaena variabilis only a one-step reduction is observed and there is no Cr(III)L attachment. From a comparison of amino acid sequences with spinach (and parsley) FdI, a likely point of Cr(III)L attachment is indicated. Comparisons are made with dithionite as reductant. In addition, square-wave voltammetry on spinach Fe(III)Fe(III).Cr(III)L gives two reduction potentials -273 and -410 mV vs NHE. The different redox products have been characterized by EPR. Using (1)H NMR line-broadening techniques, evidence for Cr(III)L binding at a surface site close to Tyr-25/Tyr-82 is obtained. Also from investigations with redox-inactive [Cr(en)(3)](3+) as a competitive inhibitor for Cr(II)L reduction of spinach Fe(III)Fe(III), Tyr-25/Tyr-82 is proposed as the site for Cr(II)L reduction. From an extension of studies to include reduction of Fe(III)Fe(III).Cr(III)L with Cr(II)L, evidence is obtained for a second reaction site when that at Tyr-25/Tyr-82 is no longer available.

Crystal Structures and Acetonitrile Exchange of Palladium(II) Complexes with Bis(3-aminopropyl)amine and Diethylenetriamine. Effects of Ion-Pair Formation

Abstract The crystal structures of palladium(II) complexes with tridentate amine and acetonitrile, [Pd(CH3CN)(dptn)](CF3SO3)2 (1) and [Pd(CH3CN)(dien)](CF3SO3)2 (2) (dptn = bis(3-aminopropyl)amine and dien = diethylenetriamine), have been determined by X-ray crystal structure analysis. The central palladium(II) ion has a distorted square-planar geometry. The five-membered chelate rings of the dien ligand in 2 are more strained than the six-membered chelate rings of the dptn ligand in 1, as indicated from the difference in the angles of terminal N–Pd–terminal N and the averaged Pd–N–C angles around the terminal nitrogens. The kinetics of acetonitrile exchange of 1, 2, and [Pd(CH3CN)(dien)](BF4)2 (3) have been investigated by the 1H NMR line-broadening technique. The observed rate constants can be expressed by kobs = k1 + k2 [CH3CN] from the dependence of kobs on the acetonitrile concentration. It has been indicated that the k1 and k2 paths proceed via the dissociative and associative modes of activation, respectively. The dissociative k1 path is promoted by the ion-pair formation where the bound ligand effect is enhanced by the electrostatic interaction between the counter anions and amine protons. The reaction mechanisms are discussed in terms of the steric and electronic factors of the bound ligands (dptn and dien) and the effect of the ion-pair formation with the counter anions (CF3SO3− and BF4−).

Factors influencing the stability of ATP in ternary complexes: Spectroscopic investigation of the interaction of certain biomimetic copper(II) complexe with ATP and AMP

The interaction of Cu(salgly)(H 2O) (1), Cu(sal-α-ala)(H 2O) (2), Na(salglygly)Cu(II) (3), Cu(glygly(H 2O) (4), [Cu(salen)H 2O)](ClO 4) (5), [Cu(sal-N-Meen)](ClO 4) (6), and [Cu(sal-N,N-Me 2en)(H 2O)](ClO 4) (7) with ATP and AMP has been studied using FTIR, ligand field and EPR spectroscopy, and 1H NMR line-broadening techniques. These complexes interact equatorially with ATP through the phosphate group and with AMP through N(7), forming 1:1 species and stabilizes it from dephosphorylation. The extent of interaction of the complexes with ATP decreases in the order 1 >- 2 > 7 > 3 >- 4. EPR studies also indicate the formation of ternary species involving the γ-phosphate group of ATP and N(7) of AMP. However, the preferential broadening of H(8) resonance for all the complexes indicates the binding of ATP through N(7) of the purine base moiety and possibly through phosphate also. ATP was found to be dephosphorylated by [Cu(salen)H 2O)] + and [Cu(sal-N-Meen)] + complexes. Our observations indicate that the primary amino group having the potential to form hydrogen bonds with ATP coupled with the positive charge on the chelate in addition to N(7) coordination may facilitate dephosphorylation.

Oxygen-17 Magnetic Resonance Study of the Oxygen Exchange of Methyl- and Butylarsonic Acids and Dimethylarsinic Acid with Solvent Water

Abstract The rates of the oxygen-exchange reactions of methyl- and butylarsonic acids and dimethylarsinic acid with solvent water were measured in the pH range below 6, and at temperatures between 50 and 90 °C, by using the 17O NMR line-broadening technique. For methylarsonic acid, the rate RMAA is expressed by the following rate law: RMAA = kh1[H+][H2A] + k1[H2A] + kh2[H+][H2A]2 + k2[H2A]2 + k3[H2A][HA−], where H2A and HA− designate CH3AsO3H2 and CH3AsO3H−, respectively. For butylarsonic acid, the rate law is expressed as: RBAA = kh1[H+][n–C4H9AsO3H2]. For dimethylarsinic acid, its conjugated acid is involved in the rate law as: RCAA = kh1′[(CH3)2AsO2H2+] + khh1′[H+][(CH3)2AsO2H2+]. The exchange rates of methyl- and butylarsonic acids and dimethylarsinic acid (50—90 °C) were analyzed in terms of the respective rate law to obtain the values of the rate constants involved in the rate laws. The activation parameters of these rate constants were obtained; for both the first- and second-order rate constants, the activation energies are characteristic of their small values (from 45 to 20 kJ mol−1), while the activation entropies have large negative values, which are found in the range between −92 and −145 J K−1 mol−1.

Pressure dependence of the rate constants for the electron/atom self-exchange between MII (cp2 and MIV) (cp)2 = X+ (M = ruthenium, osmium; X = bromide, iodide; cp = cyclopentadienide) as a function of solvent

The rate constants for the atom/electron self-exchange between Ru(cp) 2 and Ru(cp) 2 Br + (cp represents the cyclopentadienide anion), Ru(cp) 2 and Ru(cp) 2 I + , and Os(cp) 2 and Os(cp) 2 I + were measured by 1 H NMR line-broadening techniques as a function of pressure and solvent

Variable-temperature and -pressure multinuclear magnetic resonance study of solvent exchange at the tris(ethylenediamine)nickel(II) ion in ethylenediamine

The solvent exchange between ethylenediamine molecules in the bulk solvent and ethylenediamine molecules coordinated to the nickel(II) ion in neat ethylenediamine and mixtures involving N,N-dimethylformamide as a diluent has been studied by the 1 H, 13 C, and 14 N NMR line-broadening technique

Alkene exchange kinetics in bis(.eta.2-alkene)(2,4-pentanedionato)rhodium(I) investigated by NMR spectroscopy

The alkene exchange kinetics in bis(η 2 -alkene) (2,4-pentanedionatro) rhodium(I) has been studied by 1 H and 13 C NMR line-broadening techniques for ethylene and cis-2-butene. The ethylene exchange goes by an overall second-order reaction, first order in both complex and ethylene

Electron self-exchange study of the coordination-number-invariant pentacoordinate copper(I/II) couple [CuI,II((5-meimidh)2DAP)]+/2+

The electron self-exchange rate of the [Cu I,II ((5-MeimidH) 2 DAP)] +/2+ cation coupled has been determined in CD 3 CN, as a function of temperature, by dynamic NMR line-broadening techniques

Redox-induced arene hapticity change in a mixed-sandwich rhodium(III/II/I) system: an illustrative mechanistic application of electron-exchange kinetics

Abstract : Rate constants for homogeneous self exchange, kh(ex), and electrochemical exchange, ke(ex), have been measured for the first and second reduction steps of (eta 5-c5Me5) (eta 6-C6-Me6) M2+, where M = Rh and Co, in order to assess the manner in which the eta 6 yields eta 4 arene hapticity change observed upon formation of the Rh(I) two-electron reduction product is coupled with electron transfer. The analogous Co(III) / (II) / (I), mixed- sandwich system was chosen for comparison, because, unlike Rh(III)/(II)/(I), the arene retains the eta 6 configuration upon reduction. The kh(ex) values were evaluated in acetone, acetonitrile, and benzonitrile by utilizing the proton NMR line-broadening technique, and the ke(ex) values were obtained in these solvents and also nitrobenzene and propylene carbonate by using phase-selective ac voltammetry at an annealed gold electrode. The corresponding variations in kh(ex) and kh(ex) with the redox couple are shown to be uniformly consistent with the expectations of Marcus theory, indicating that the electrochemical as well as homogeneous-phase exchange kinetics refer to outer-sphere pathways, as desired. Both the Rh(III) / (II) and Rh(II) / (I) couples exhibit smaller rate constants in a given solvent than the corresponding cobalt systems; while these differences are mild for the former couple, the rate constants for Rh(II) / (I) are substantially smaller than for the other reactions.

Ligand-substitution and electron-transfer reactions of pentacoordinate copper(I) complexes

The kinetics of the electron self-exchange for the Cu(I)/Cu(II) couple of the pentacoordinate complex of 2,6-bis(1-((2-imidazol-4-ylethyl)imino)ethyl)pyridine ((imidH)/sub 2/DAP) has been studied in CD/sub 3/CN by dynamic NMR line-broadening techniques. The rate constant, k/prime//sub 22/ is 1.31 /times/ 10/sup 4/ M/sup /minus/1/ s/sup /minus/1/ at 25/degree/C and an ionic strength of 22.3 mM (Me/sub 4/NBF/sub 4/). Correction for ion pairing gives k''/sub 22/ = 2.8 /times/ 10/sup 4/M/sup /minus/1/ s/sup /minus/1/ at /mu/ = 38 mM and 25/degree/C, which is a factor of 10 greater than that for the (Cu((py)/sub 2/DAP))/sup n+/ analogue. With the use of these two self-exchange rate constants, the Marcus cross relationship accurately predicts the measured cross-exchange rate constant. Transfer of the ligand from (Cu/sup I/((py)/sub 2/DAP))/sup +/ to (Zn/sup II/(CH/sub 3/CN)/sub 6/)/sup 2+/ at /mu/ = 20 mM was investigated at 25/degree/C in CH/sub 3/CN by using the stopped-flow technique. With excess Zn(II), saturation kinetics were observed; this behavior is attributed to rate-limiting dissociation of the copper complex with a rate constant of 310 s/sup /minus/1/. These results are considered in evaluating the possibility of an inner-sphere mechanism of electron transfer. 33 refs., 6 figs., 3 tabs..

Fructose 1,6-bisphosphate: isomeric composition, kinetics, and substrate specificity for the aldolases.

13C NMR shows fructose 6-phosphate and fructose 1,6-bisphosphate to contain respectively 4.1 and 2.0% keto isomer at room temperature. The lower value for fructose 1,6-bisphosphate can be attributed to the electron-withdrawing effect of the C-1 phosphate. Measurements of the ring-opening rates of the alpha and beta anomers of fructose, 1,6-bisphosphate by an NMR line-broadening technique show them to be about 8 and 35 S-1, respectively, at pH 7.2, and 25degreesC. The value for the predominant beta anomer is threefold greater than the turnover rate of muscle aldolase so that, if the kinetic properties of the keto form were favorable, the reaction could proceed entirely through the keto form in solution. The kinetic properties of a fructose 1,6-bisphosphate(keto) analogue, 5-deoxyfructose, 1,6-bisphosphate, in the muscle aldolase reaction are more favorable (Vmax = 2.6, Km = 0.11 X 10(-6) M) than those of fructose 1,6-bisphosphate total (Vmax = 1, Km = 2.3 X 10(-6)M), giving a value of Vmax/Km that is 56 times greater for the 5-deoxy analogue. At the 2.0% concentration of the keto form this is sufficient to account for the steady-state rate and requires that the beta form, present at 40 times greater concentration, contributes little to the cleavage rate. With yeast aldolase the cleavage rate can be explained by the rapid spontaneous ring opening and reaction of the keto form with the enzyme. In view of the high rate of ring opening and the excellent properties of the keto form, previous rapid kinetic studies favoring action of cyclic forms may require reevaluation.