pH-Dependent Change in Km Values and Translocation Constants of the Uptake System (original) (raw)
Related papers
The Journal of General Physiology, 1974
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 (Km 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 Vmax values at low substrate concentration versus proton concentration results in a Km for protons of 0.14 µM, 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...
Buffer-facilitated proton transport. pH profile of bound enzymes
Biochimica et Biophysica Acta (BBA) - Enzymology, 1974
In heterogeneous systems conjugate acid base pairs, besides their conventionally accepted static role as buffers, can also play a dynamic role in facilitating proton transport. When protons are generated and consumed at different locations, the conjugate base binds the proton at the "source" and the resulting acid diffuses to the "sink". 2. Both the buffering capacity of an acid base pair and its ability to facilitate proton transport are maximum when its pKa is close to the pH of the medium. The transport facilitating effect can be appreciable at much lower concentrations than those needed for buffering capacity. 3. The effect of proton transport has been theoretically investigated by using a model consisting of a H-producing enzymic surface reaction, which occur with bound enzymes both in vitro and in vivo. 4. The enhancement of proton transport is quantitatively expressed by the extent of transport facilitation, ~:. The interplay of facilitated proton diffusion and reaction kinetics is quantified by the proton modulus,/z. 5. At a given pKA the magnitude of transport facilitation increases with the acid-base concentration and the pH of the solution. The pKa value plays a dual role in determining both the concentration of available carriers and the affinity of the proton to the carrier. 6. Due to its high proton affinity OH can also facilitate proton transport already at pH 7, but in the presence of buffers its effect becomes significant only at higher pH values. 7. When the extent of transport facilitation sharply increases with the pH of the substrate solution, at relatively low buffer concentrations bound enzymes display sigmoidal pH profiles with sharp peaks. Previously reported experimental data with membrane bound enzymes can be interpreted in view of these theoretical findings. 8. It is postulated that buffers ubiquitous in biochemical and living systems can play an important role also by facilitating proton transport.
The pre-steady-state kinetics of conformational changes in sugar transporters
Biochemical Society transactions, 1994
Biochemical Society Transactions mine whether these residues play a role in charge transfer. In rabbit SGLT1 we have mutated Asp-176 to asparagine and to alanine and measured charge transfer. Dramatic changes occurred with the Aspl76Ala mutant, i.e. V,,,s decreased from + 1 to-46 mV and the z decreased with depolarization instead of increasing. This result alone would suggest that Asp-176 is a charged residue directly involved in sensing the membrane field, but the Asp 176Asn mutation only produced modest changes in charge-transfer kinetics. W e conclude that polar residues, and not necessarily charged residues, constitute the charge sensor in SGLT1. W e are presently extending our studies to other cloned transporters. In general, the results so far indicate that the steady-state and pre-steadystate kinetics of Na+-Ins, H+-glucose and Na+-CI-taurine cotransporters have much in common with SGLT1.
Biochemistry, 1994
The D-xylose isomerase of Streptomyces olivochromogenes is a Mg2+-or Mn2+-dependent enzyme that catalyzes the aldose-ketose isomerization of xylose to xylulose or of glucose to fructose. Proton exchange into water during enzyme-catalyzed isomerization of C-2 tritiated glucose at 15,25 and 55 " C shows <0.6% exchange (the loss of one proton in every billion turnovers). High concentrations of guanidine hydrochloride and extremes of p H had no effect on the amount of exchange detected. Such a low percentage of exchange is inconsistent with a proton-transfer mechanism as the main kinetic pathway for isomerization. 19F N M R experiments showed no release of fluoride after incubation of the enzyme for 4 weeks with 800 m M 3-deoxy-3-fluoroglucose or 3-deoxy-3-fluoroallose (both are competitive inhibitors with Ki values of 600 mM). This result is also inconsistent with a proton-transfer mechanism. A hydride-shift mechanism following ring opening has been proposed for the isomerization. Enzyme-catalyzed ring opening was directly measured by demonstrating HIS release upon reaction of xylose isomerase with 1-thioglucose. D-Xylose isomerasecatalyzed interconversion of glucose to fructose exhibited linear Arrhenius behavior with an activation energy of 14 kcal/mol from 0 to 50 OC. No change in rate-determining step occurs over this temperature range. 13C N M R experiments with glucose show that enzyme-bound magnesium or manganese does not interact specifically with any one site on the sugar. These results are consistent with nonproductive binding modes for the substrate glucose in addition to productive binding.
1993
A quantitative analysis of the proton movements associated with the initial uptake rate of weak short-chain carboxylic acids was developed in order to estimate proton/carboxylate symports stoichiometries. The yeast Candida utilis was used as a biological model and the deduced equations were applied on the elucidation of the proton/carboxylate symports stoichiometries of lactate, succinate and citrate in a strain of that yeast species at different pH values. At pH 5.0, the proton/lactate and the proton/succinate symport stoichiometry was 1:1. In the cases of the proton/lactate and proton/citrate symports it appears that the stoichiometry ratio increased with increasing extracellular pH.
Proton-linked D-xylose transport in Escherichia coli
Journal of Bacteriology, 1980
The addition of xylose to energy-depleted cells of Escherichia coli elicited an alkaline pH change which failed to appear in the presence of uncoupling agents. Accumulation of [14C]xylose by energy-replete cells was also inhibited by uncoupling agents, but not by fluoride or arsenate. Subcellular vesicles of E. coli accumulated [14C]xylose provided that ascorbate plus phenazine methosulfate were present for respiration, and this accumulation was inhibited by uncoupling agents or valinomycin. Therefore, the transport of xylose into E. coli appears to be energized by a proton-motive force, rather than by a phosphotransferase or directly energized mechanism. Its specificity for xylose as inducer and substrate and the genetic location of a xylose-H+ transport-negative mutation near mtl showed that the xylose-H+ system is distinct from other proton-linked sugar transport systems of E. coli.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1993
A quantitative analysis of the proton movements associated with the initial uptake rate of weak short-chain carboxylic acids was developed in order to estimate proton/carboxylate symports stoichiometries. The yeast Candida utilis was used as a biological model and the deduced equations were applied on the elucidation of the proton/carboxylate symports stoichiometries of lactate, succinate and citrate in a strain of that yeast species at different pH values. At pH 5.0, the proton/lactate and the proton/succinate symport stoichiometry was 1:1. In the cases of the proton/lactate and proton/citrate symports it appears that the stoichiometry ratio increased with increasing extracellular pH.
Evidence for a proton/sugar symport in the yeast Rhodotorula gracilis (glutinis).
Biochemical Journal, 1978
1. The uptake of monosaccharides and polyols in the obligatory aerobic yeast Rhodotorula gracilis (glutinis) was accompanied by proton uptake. 2. The halfsaturation constant of transport, KT, depended on pH, changing from about 2mM at pH4.5 to 80mM at pH8.5 for D-xylose; this change of the effective carrier affinity was reversible. 3. The apparent dissociation constant of the monosaccharide carrier was estimated at pKa 6.75. 4. At pH8.5, when the pH gradient across the cell membrane vanished, no sugar accumulation was demonstrable. 5. The half-saturation constants of sugar uptake and H+ co-transport were very similar to each other, the latter obviously being controlled by the former. 6. The H+/sugar stoicheiometry remained constant under various physiological conditions; it amounted to one H+ ion per sugar molecule taken up. 7. The data are interpreted as a strong piece of evidence in favour of the active monosaccharide transport in R. gracilis (glutinis) being an H+-symport energized by the electrochemical gradient of H+ across the plasma membrane of the yeast.
In Silico Kinetic Study of the Glucose Transporter
Journal of Biological Physics, 2007
Glucose transport in plasma membranes is the prototypic example of facilitated diffusion through biological membranes, and transport in erythrocytes is the most widely studied. One of the oldest and simplest models describing the kinetics of the transport reaction is that of alternating conformers, schematized in a cycle of four partial reactions where glucose binds and dissociates at two opposite steps, and the transporter undergoes transconformations at the other two opposite steps. The transport kinetics is entirely defined by the forward and backward rate constants of the partial reactions and the glucose and transporter concentrations at each side of the membrane, related by the law of mass action. We studied, in silico, the effect of modifications of the variables on the transient kinetics of the transport reaction. The simulations took into account thermodynamic constraints and provided results regarding initial velocities of transport, maximal velocities in different conditions, apparent influx and efflux affinities, and the turnover number of the transporter. The results are in the range of those experimentally reported. Maximal initial velocities are obtained when the affinities of the ligand for the transporter are the same at the extraand intracellular binding sites and when the equilibrium constants of the transconformation steps are equal among them and equal to 1, independently of the obvious effect of the increase of the rate constant values. The results are well adjusted to Michaelis-Menten kinetics. A larger initial velocity for efflux than for uptake described in human erythrocytes is demonstrated in a model with the same dissociation constants at the outer and inner sites of the membrane. The larger velocities observed for uptake and efflux when transport occurs towards a glucose-containing trans side can also be reproduced with the alternating conformer model, depending on how transport velocities are measured.