Buffer-facilitated proton transport. pH profile of bound enzymes (original) (raw)
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pH dependence of proton translocation by Escherichia coli
The Journal of biological chemistry, 1992
Proton translocation in spheroplasts from Escherichia coli has been studied in two mutants, one of which expresses cytochrome o and the other cytochrome d as the terminal oxidase. Using the O2 pulse method, the H+/e- ratio of proton translocation associated with cytochrome o was confirmed to be near 2 at neutral pH, but was found to decrease considerably when the medium pH was raised above 8. At high pH there was an increase in H+/OH- permeability of the cell membrane, but this was not sufficient to explain the decline in proton ejection. The pH effect was confined to cytochrome o-linked activity. It was not present when cytochrome d generated the electrochemical proton gradient. This makes it improbable that the Na+/H+ antiporter is responsible. The most likely explanation for our finding is that there is a "slip" in the proton-pumping mechanism of cytochrome o at high pH.
Bioelectrochemistry and Bioenergetics, 1998
The widespread belief that the electric potential difference Df across membranes with electrogenic proton transport varies nearly m Ž. linearly with the pH difference D pH upon build-up of the proton-motive force is critically examined. First, we analyse experimental literature data concerning rat liver and yeast mitochondria, and E. coli. We then present a model describing the interrelation between the D pH and Df as the activities of the proton pumps or H q-ATPase or the influence of the proton leak vary. It is based on the m quasi-electroneutrality condition, the dissociation equilibrium of impermeant weak acids, a simple description of the cation-proton antiporters and cation leak, and the Nernst equation applied to all those ions subsisting in equilibrium. The model yields a nonlinear equation giving Df as a function of D pH. In various situations this function is quasi-linear in physiologically relevant ranges of D pH. m Thus, the linearity hypothesis can be substantiated theoretically, but is not necessarily justified under all circumstances. It is shown that Ž the slope of the Df vs. D pH curve is, in the quasi-linear regions, about y2.303 RTrF thus having the same value, but the opposite m. sign as in the Nernst equation when the cation-proton antiporters are absent or completely inhibited, and can be much higher in absolute value when these antiporters are operative.
Biochemical Journal, 2005
PAT1 is a recently identified member of the PAT family of proton/ amino acid co-transporters with predominant expression in the plasma membrane of enterocytes and in lysosomal membranes of neurons. Previous studies in Xenopus oocytes expressing PAT1 established proton/substrate co-transport associated with positive inward currents for a variety of small neutral amino acids. Here we provide a detailed analysis of the transport mode of the murine PAT1 in oocytes using the two-electrode voltage-clamp technique to measure steady-state and pre-steady-state currents. The GPC (giant patch clamp) technique and efflux studies were employed to characterize the reversed transport mode. Kinetic parameters [K m (Michaelis constant) and I max (maximum current)] for transport of various substrates revealed a dependence on membrane potential: hyperpolarization increases the substrate affinity and maximal transport velocity. Proton affinity for interaction with PAT1 is almost 100 nM, corresponding to a pH of 7.0 and is independent of substrate. Kinetic analysis revealed that binding of proton most likely occurs before substrate binding and that the proton and substrate are translocated in a simultaneous step. No evidence for a substrate-uncoupled proton shunt was observed. As shown by efflux studies and current measurements by the GPC technique, PAT1 allows bidirectional amino acid transport. Surprisingly, PAT1 exhibits no pre-steady-state currents in the absence of substrate, even at low temperatures, and therefore PAT1 takes an exceptional position among the ion-coupled co-transporters.
Chemical Engineering Science, 1984
The performance of immobilized enzymes which catalyse H + ion generating reactions is markedly al&ted by diffusional limitations on H+ ions. Addition of small amounts of weak acids can facilitate the rate of removal of these protons from within the porous interstices of the enzyme support, by providing a parallel path for them. The importance of this transport facilitating role of weak acids on the performance of immobilized enzymes is examined theoretically for the case of internal (pore) diffusion. The effect of these weak acids can conveniently be lumped into an apparent diffusion coel%cient (DW) of the protons. This diffusion coefficient depends on the concentration of H+ ions, the pK of the weak acid and its total concentration, C,. For given values of C, and pK, the plot of Dapp vs pH has a sigmoidal shape, with a very steep variation near the pK of the weak acid. For any particular weak acid (i.e. a given pK), an increase in its total concentration, C, results in a monotonic increase in DaP. Even at values of CT as low as IO-'M, for pH values larger than pK, D is many orders of magnitude greater than the true diffusivity of H+ ions. The above steep increase o$D mechanism of the heterogenous reaction from a regime where "L to very large values can transform the th diffusion and kinetics play a role to one which is entirely controlled by kinetics, if the Thiele modulus, r$, is not too large (less than about 5 for C, = 10-s M). This suggests that it is possible to maximize the activity of the immobilized enzyme by a proper choice of the pH of the bulk solution and the type of weak acid: The pH in the bulk solution should be close to the optimum pH of the soluble enzyme, and the weak acid should be in sufficient amount and have a pK close to the above optimum pH. If the bulk pH is larger than the above optimum, a certain amount of diffusional resistance in the pellet is necessary to achieve maximum activity. Such a control can be achieved by using an optimum amount of weak acid. Detailed calculations have been carried out, to determine the activity vs bulk pH and the activity vs bulk concentration profiles for various values of the Thiele modulus, when the substrate concentration is small as well as when it is large in comparison with the Michaelis constant.
Proton Transport via the Membrane Surface
Biophysical Journal, 2002
Some proton pumps, such as cytochrome c oxidase (C c O), translocate protons across biological membranes at a rate that considerably exceeds the rate of proton transport to the entrance of the proton-conducting channel via bulk diffusion. This effect is usually ascribed to a proton-collecting antenna surrounding the channel entrance. In this paper, we consider a realistic phenomenological model of such an antenna. In our model, a homogeneous membrane surface, which can mediate proton diffusion toward the channel entrance, is populated with protolytic groups that are in dynamic equilibrium with the solution. Equations that describe coupled surface-bulk proton diffusion are derived and analyzed. A general expression for the rate constant of proton transport via such a coupled surface-bulk diffusion mechanism is obtained. A rigorous criterion is formulated of when proton diffusion along the surface enhances the transport. The enhancement factor is found to depend on the ratio of the surface and bulk diffusional constants, pK a values of surface protolytic groups, and their concentration. A capture radius for a proton on the surface and an effective size of the antenna are found. The theory also predicts the effective distance that a proton can migrate on the membrane surface between a source (such as CcO) and a sink (such as ATP synthase) without fully equilibrating with the bulk. In pure aqueous solutions, protons can travel over long distances (microns). In buffered solutions, the travel distance is much shorter (nanometers); still the enhancement effect of the surface diffusion on the proton flow to a target on the surface can be tens to hundreds at physiological buffer concentrations. These results are discussed in a general context of chemiosmotic theory.
pH-Dependent Change in Km Values and Translocation Constants of the Uptake System
2013
The proton concentration in the medium affects the maximal velocity of sugar uptake with a Km of 0.3 mM (high affinity uptake). By decreasing the proton concentration a decrease in high affinity sugar uptake is observed, in parallel the activity of a low affinity uptake system (K, of 50 mM) rises. Both systems add up to 100 %. The existence of the carrier in two conformational states (protonated and unprotonated) has been proposed therefore, the protonated form with high affinity to 6-deoxyglucose, the unprotonated form with low affinity. A plot of extrapolated V. values at low substrate concentration versus proton concentration results in a Km for protons of 0.14 AiM, i.e. half-maximal protonation of the carrier is achieved at pH 6.85. The stoichiometry of protons cotransported per 6-deoxyglucose is close to 1 at pH 6.0-6.5. At higher pH values the stoichiometry continuously decreases; at pH 8.0 only one proton is cotransported per four molecules of sugar. Whereas the translocation...
Elucidation of the Proton Transport Mechanism in Human Carbonic Anhydrase II
Journal of the American Chemical Society, 2009
Human carbonic anhydrase II (HCA II) is one of the fastest known enzymes, which utilizes a ratelimiting proton transport (PT) step in its enzymatic reaction. To evaluate the PT event at an atomistic level, the multistate empirical valence bond (MS-EVB) method has been utilized in this work. It is observed that the PT event in HCA II exploits a transient active site water cluster to transport the excess proton between the catalytic zinc-bound water/hydroxide and the proton shuttling residue, His64. This PT event is found to be dependent on the enzyme's ability to form and stabilize the active site water cluster in addition to its ability to orient His64 in a favorable conformation. Evaluation of the PT free energy barrier for different orientations of His64 reveals this residue's vital role as a proton transporter and elucidates its direct effect on the barrier to PT through the active site water. It is suggested that the rate-limiting step oscillates between the active site water PT event to His64 and the de/protonation of His64 depending on the exogenous buffer concentration and the orientation of His64. In the absence of a PT acceptor/donor at position 64, it is found that the excess proton will utilize one of three distinct paths to enter/leave the active site. This latter result not only allows for an increased understanding of how enzymes capitalize on the protein/solvent interface to guide excess protons to/from areas of interest, it also provides valuable insight into the chemical rescue experiments on HCA II mutants.
Biophysical Journal, 2004
We have developed a mathematical model in concert with an assay that allows us to calculate proton (H 1 ) flux and conductance through a single F o of the F 1 F o ATP synthase. Lipid vesicles reconstituted with just a few functional F o from Escherichia coli were loaded with 250 mM K 1 and suspended in a low K 1 solution. The pH of the weakly buffered external solution was recorded during sequential treatment with the potassium ionophore valinomycin, the protonophore carbonyl cyanide 3-chlorophenylhydrazone, and HCl. From these pH traces and separate determinations of vesicle size and lipid concentration we calculate the proton conductance through a single F o sector. This methodology is sensitive enough to detect small (15%) conductance changes. We find that wild-type F o has a proton flux of 3100 6 500 H 1 /s/F o at a transmembrane potential of 106 mV (25°C and pH 6.8). This corresponds to a proton conductance of 4.4 fS.