Solvent isotope effects on alpha-glucosidase

The solvent kinetic isotope effects (SKIE) on the yeast alpha-glucosidase-catalyzed hydrolysis of p-nitrophenyl and methyl-d-glucopyranoside were measured at 25 degrees C. With p-nitrophenyl-D-glucopyranoside (pNPG), the dependence of k(cat)/K(m) on pH (pD) revealed an unusually large (for glycohydrolases) solvent isotope effect on the pL-independent second-order rate constant, (DOD)(k(cat)/K(m)), of 1.9 (+/-0.3). The two pK(a)s characterizing the pH profile were increased in D(2)O. The shift in pK(a2) of 0.6 units is typical of acids of comparable acidity (pK(a)=6.5), but the increase in pK(a1) (=5.7) of 0.1 unit in going from H(2)O to D(2)O is unusually small. The initial velocities show substrate inhibition (K(is)/K(m) approximately 200) with a small solvent isotope effect on the inhibition constant [(DOD)K(is)=1.1 (+/-0.2)]. The solvent equilibrium isotope effects on the K(is) for the competitive inhibitors D-glucose and alpha-methyl D-glucoside are somewhat higher [(DOD)K(i)=1.5 (+/-0.1)]. Methyl glucoside is much less reactive than pNPG, with k(cat) 230 times lower and k(cat)/K(m) 5 x 10(4) times lower. The solvent isotope effect on k(cat) for this substrate [=1.11 (+/-0. 02)] is lower than that for pNPG [=1.67 (+/-0.07)], consistent with more extensive proton transfer in the transition state for the deglucosylation step than for the glucosylation step.     

Solvent isotope effects on microtubule polymerization and depolymerization

The initial velocity of polymerization of purified beef brain tubulin has been determined at various values of pH or pD in water and in H₂O-D₂O mixtures. D₂O was shown to inhibit both polymerization at 37 °C and depolymerization measured at 5 °C and 37 °C. The microtubules formed in D₂O were indistinguishable from those formed in H₂O, by electron microscope examination. In 93% D₂O the pL²versus rate of polymerization curve was displaced about one unit towards higher pL values. In certain regions of the pL versus rate curve, a stimulation in the rate of polymerization by D₂O is observed. The extent of polymerization at the optimum pL value was not affected by D₂O.     

Solvent isotope effects on the reaction catalyzed by yeast hexokinase

The pH dependence of the maximum velocity (V) for the phosphorylation of glucose, the V/Kglucose and the V/KMgATP have been obtained in H2O and 2H2O. In H2O, V decreases below a pK of 5.8, V/Kglucose decreases below a pK of 6.1 and V/KMgATP decreases below a pK of 6.7. In 2H2O, complex behavior is observed for these parameters as a function of pD. The ratios of the parameters in H2O and 2H2O above their respective pK values give solvent deuterium isotope effects of about 1.5-1.7 for all three parameters. When 1,5-anhydromannitol is used as an alternative substrate, an isotope effect different than unity is obtained only for V/K1,5-anhydromannitol which gives a value of about 0.7. Both the complex pH profiles and the relative magnitude of the isotope effects are interpreted in terms of a pH-dependent change in the E X glucose complex.     

Solvent isotope effects on the refolding kinetics of hen egg-white lysozyme.

Solvent isotope effects have been observed on the in vitro refolding kinetics of a protein, hen lysozyme. The rates of two distinct phases of refolding resolved by intrinsic fluorescence have been found to be altered, to differing extents, in D2O compared with H2O, and experiments have been conducted aimed at assessing the contributions to these effects from various possible sources. The rates were found to be essentially independent of whether backbone amide nitrogens were protiated or deuterated, indicating that making and breaking of their hydrogen bonding interactions is not associated with a substantial isotope effect. Neither were the rates significantly affected by adding moderate concentrations of sucrose or glycerol to the refolding buffer, suggesting that viscosity differences between H2O and D2O are also unlikely to explain the isotope effects. The data suggest that different factors, acting in opposing directions, may be dominant under different conditions. Thus, the isotope effect on the rate-determining step was found to be qualitatively reversed on going to low pH, suggesting that one component is probably associated with changes in the environments of carboxylate groups in forming the folding transition state. This term disappears at low pH as these groups are protonated and an opposing effect then dominates. It was not possible to identify this other effect on the basis of the present data, but a dependence of the hydrophobic interaction on solvent isotopic composition is a likely candidate.     

Solvent isotope and viscosity effects on the steady-state kinetics of the flavoprotein nitroalkane oxidase

The flavoprotein nitroalkane oxidase catalyzes the oxidative denitrification of a broad range of primary and secondary nitroalkanes to yield the respective aldehydes or ketones, hydrogen peroxide and nitrite. With nitroethane as substrate the ^D2O(k_cat/K_M) value is 0.6 and the ^D2Ok_cat value is 2.4. The k_cat proton inventory is consistent with a single exchangeable proton in flight, while the k_cat/K_M is consistent with either a single proton in flight in the transition state or a medium effect. Increasing the solvent viscosity did not affect the k_cat or k_cat/K_M value significantly, establishing that nitroethane binding is at equilibrium and that product release does not limit k_cat.     

Solvent isotope effects in intramolecular catalysis: Acyl transfer reactions of 4-nitrophenyl 5-nitrosalicylate in aqueous tris buffer

A kinetic study of the reactions of 4-nitrophenyl 5-nitrosalicylate in aqueous Tris buffer at 25°C has revealed three kinetically significant reactions: Tris aminolysis of the un-ionized substrate, Tris aminolysis of the ionized substrate, and spontaneous hydrolysis of the ionized substrate. Solvent isotope effects have been measured for all three reactions. Mechanisms are discussed, and the conclusion is reached that intramolecular catalysis is operating in only the spontaneous hydrolysis.     

Solvent effects on the structure,dynamics and activity of lysozyme

Kuntz and Kauzmann have argued that dehydrating a protein results in conformational changes. In contrast, Rupleyet al. have developed a hydration model which involves no significant change in conformation; the onset of enzyme activity in hen egg-white lysozyme at hydration values of about 0.2 g water/g protein they ascribe rather to a solvation effect. Using a direct difference infra-red technique we can follow specific hydration events as water is added to a dry protein. Conformational studies of lysozyme using laser Raman spectroscopy indicate changes in conformation with hydration that are complete just before measurable activity is found. Parallel nuclear magnetic resonance measurements of exchangeability of the main chain amide hydrogens, as a function of hydration from near dryness, suggest a hydration-related increase in conformational flexibility which occurs before-and is probably necessary for-the Raman-detected conformational changes. Very recent inelastic neutron scattering measurements provides direct evidence of a flexibility change induced by hydration, which is apparently necessary before the enzyme can achieve adequate flexibility for it to begin to function.     

Effect of crosslinking on mitochondrial structure and function

Rat liver mitochondria were treated with ethylacetimidate

1 Abbreviations: SDS, sodium dodecylsulfate; DMS, dimethylsuberimidate; EA, ethylacetimidate; MBI, methylbutyrimidate; TMPD, N, N′, N′-tetramethyl-p-phenylenediamine.

and methylbutyrimidate, monofunctional imidates, and with dimethylsuberimidate, a bifunctional imidate, and the effects on structure and function studied. Mitochondria treated with 5 mM dimethylsuberimidate or greater did not respond osmotically when placed in deionized water. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that at concentrations > 5 mM dimethylsuberimidate nearly all mitochondrial polypeptides failed to enter 6% gels, indicating crosslinking of both membrane and soluble proteins. Extensive amidination by ethylacetimidate and methylbutyrimidate had little effect on ascorbate-tetramethylphenylenediamine oxidase while extensive inhibition resulted from dimethylsuberimidate treatment. The possible involvement of molecular motion in electron transport is discussed.     

Multiple effects of DiS-C3(5) on mitochondrial structure and function.

3,3'-Dipropyl-2,2'-thiadicarbocyanine iodide [DiS-C(3)(5)], often used as a tracer dye to assess the mitochondrial membrane potential, was investigated in detail regarding its effects on the structure and function of isolated mitochondria. As reported previously, DiS-C(3)(5) had an inhibitory effect on NADH-driven mitochondrial electron transfer. On the contrary, in the presence of inorganic phosphate, DiS-C(3)(5) showed dose-dependent biphasic effects on mitochondria energized by succinate. At higher concentrations, such as 50 micro m, DiS-C(3)(5) accelerated mitochondrial oxygen consumption. Measurements of the permeability of DiS-C(3)(5)-treated mitochondrial membranes to poly(ethylene glycol) and analysis of mitochondrial configuration by transmission electron microscopy revealed that the accelerating effect of DiS-C(3)(5) on mitochondrial oxygen consumption reflects the induction of the mitochondrial permeability transition (PT). When the mitochondrial PT was induced by DiS-C(3)(5), release of mitochondrial cytochrome c was observed, as in the case of the PT induced by Ca(2+). On the contrary, at a low concentration such as 5 micro m, DiS-C(3)(5) showed an inhibitory effect on the latent oxygen consumption by mitochondria. This effect was shown to reflect inhibition of the PT induced by a low concentration of Ca(2+). Furthermore, in the absence of inorganic phosphate, DiS-C(3)(5) caused mitochondrial swelling. Under this condition, DiS-C(3)(5) caused changes in the membrane status of the mitochondria, but did not induce a release of mitochondrial cytochrome c.     

Solvent isotope effects and the pH dependence of laccase activity under steady-state conditions

Solvent isotope effects and the pH dependence of laccase catalysis under steady-state conditions were examined with a rapid reductant to assess the potential roles of protein protic groups and the catalytic mechanism. The pH dependence of both reductant-dependent and reductant-independent steps showed bell-shaped profiles implicating at least two protic groups in each case. The apparent pKa values were: for the reductant-independent step(s), pK alpha 1 = 8.98 +/- 0.02 and pK alpha 2 = 5.91 +/- 0.03; for the reductant-dependent step(s), pK' alpha 1 = 7.55 +/- 0.12, pK' alpha 2 = 8.40 +/- 0.23. No solvent isotope effect on reductant-dependent steps was detected other than a standard shift effect. However, a significant solvent isotope effect on a reductant-independent step(s) was observed; kH/kD = 2.12 at the pH optimum of 7.5. The concentration dependence of the D2O effect indicated that a single proton was involved. Simulations of the p(H,D) data suggested that the solvent isotope effect was associated with the protein protic group required in its undissociated form (pK alpha 2). The pH effects on reductant-dependent steps are apparently associated with reductant-dependent steps that occur between O2 binding and water formation in the catalytic reaction sequence.     

The effects of manganese deficiency during prenatal and postnatal development on mitochondrial structure and function in the rat

The influence of manganese deficiency on liver trace element concentration, MnSOD activity, and mitochondrial structure and function during postnatal development was determined in rats. In both normal and manganese-deficient animals, liver manganese concentration increased with time, but in deficient rats liver manganese was lower than in controls at all ages measured. At 9 mo of age, liver manganese concentration in the deficient rats was only 20% that of controls. The developmental pattern observed for MnSOD paralleled that of liver manganese concentration in normal and deficient rats; it was lower than in controls on days 20 and 60. However, at 9 mo of age, MnSOD levels were similar in the two groups. Although there were no differences at 9 mo of age in MnSOD activity between the groups, manganese-deficient rats showed mitochondrial abnormalities in liver. Despite mitochondrial abnormalities, however, oxygen uptake and P/O ratios were normal. We suggest that the mitochondrial damage apparent at 9 mo of age is, at least in part, the result of lower than normal MnSOD activity occurring earlier. The functional significance of the abnormalities remains to be established.     

Alpha-secondary tritium isotope effects in the aqueous hydrolysis of glycopyranosides of N-acetyl-beta-D-glucosamine

Secondary tritium isotope effects were used to study the aqueous hydrolysis of a series of α- and β-glycopyranosides of N-acetyl-d-glucosamine. The magnitude of the secondary tritium isotope effects, and their dependence on the structure of the aglycone, are compatible with the carbonium ion mechanism suggested for the specific acid catalysis of these compounds by Piszkiewicz and Bruice (1–3). The secondary tritium isotope effect determined for the spontaneous hydrolysis of the p-nitrophenyl-2-acetamido-2-deoxy-β-d-glucopyranoside is not consistent with an intramolecular, nucleophilic displacement mechanism. A mechanism involving the equilibrium formation of a carbonium ion-anion pair is proposed. The relevance of these model studies to hydrolysis of oligosaccharides of N-acetyl-d-glucosamine by lysozyme is discussed.