Reaction engineering analysis of L-lysine transport by Corynebacterium glutamicum

To identify potential L-lysine export limitations by Corynebacterium glutamicum in the L-lysine production process, the excretion of L-lysine was studied in continuous and fed-batch operated stirred tank reactors. A structured biochemical model of the L-lysine excretion mechanism was used to determine the activity of the export carrier and to calculate a cell-specific concentration of the export carrier. For the biochemical characterization of this specific carrier concentration a standardized L-lysine efflux test was developed. Carrier activity, cell-specific carrier concentration, and the specific L-lysine export rate were identified as a function of pH value and L-lysine concentration in the reactors. Also, the correlation of these parameters to the metabolic state of C. glutamicum was determined. The pH value in the reactor governs the carrier activity (maximum at pH 6.5) and the specific carrier concentration (maximum at pH 8.0). The specific L-lysine export rate, as the product of carrier activity and specific carrier concentration, revealed a maximum at pH 7.0. Decreasing L-lysine productivities also correlated with decreasing specific carrier concentrations. The L-lysine concentration in the reactor had no effect on the specific carrier concentration but strongly inhibited the carrier activity. The specific export rate was reduced to 50% at 400 mM L-lysine compared to the specific export rate at 80 mM L-lysine. (c) 1996 John Wiley & Sons, Inc.     

Transient currents carried by the uncoupler,carbonyl cyanide m-chlorophenylhydrazone

The weak acid uncoupler, carbonyl cyanide m-chlorophenylhydrazone, carries protons across lipid membranes. As predicted by the carrier model, at low pH, the current changes immediately following a jump in applied potential and then remains constant. By contrast at high pH, the currents relax from an initial value to a lower value as the carrier anions redistribute in the membrane. These relaxations are slower than those seen with other lipid-soluble anions which presumably explains why they had not been detected previously.     

pH gradients in immobilized amidases and their influence on rates and yields of beta-lactam hydrolysis

The pH gradients developing within immobilized biocatalysts during hydrolysis of penicillin G and glutaryl-7-aminocephalosporanic acid have been estimated both theoretically and experimentally. For the latter a fluorimetric method for the direct measurement of the average pH value within the carrier during reaction has been developed using the pH-dependent fluorescence intensity of an enzyme-bound fluorophore determined with a fiber bundle. The theoretical calculations were based on a model for the hydrolysis with immobilized enzymes using a kinetic expression with five pH-dependent, measurable kinetic and equilibrium constants. The transport reaction differential equation which considers the laminar boundary layer has been solved numerically for the key component. The calculated values agreed well with the experimental data. Under the typical reaction conditions of penicillin G hydrolysis the average pH value in the carrier was 1 and 2.5 pH units below the bulk pH (=8) with and without buffer, respectively. The corresponding changes for the hydrolysis of glutaryl-7-aminocephalosporanic acid at bulk pH 8 in the presence of buffer was 0.5. This demonstrates the existence of considerable pH gradients in carriers during hydrolytic reactions, even in buffered systems with negligible mass transfer resistance. The low pH value causes suboptimal reaction rates, reduced equilibrium conversion, and reduced enzyme stability. These pH gradients can be minimised by using buffers with pK values approximately equal to the bulk pH used for the hydrolysis. The prediction quality of the model has been tested applying it to fixed bed reactor design. The reduction in rate and yield due to concentration and pH gradients can be overcome with simple measures such as high initial pH value and pH adjustments in segmented or recycling fixed bed reactors. Thus, enzymatic conversions with high yield and high operational effectiveness are achieved.     

Isoelectric focusing in immobilized pH gradients in the pH 10-11 range

With the synthesis of a new, strongly basic Immobiline (pK 10.3 at 10 degrees C) it has been possible to formulate a new pH 10-11 recipe for focusing very alkaline proteins, not amenable to fractionation with conventional isoelectric focusing in carrier ampholyte buffers. In this formulation, water is added as an acidic Immobiline having pK = 14 and a unit molar concentration (or with a pK = 15.74 and standard 55.56 molarity) since around pH 11 its buffering power becomes significant. The gel contains a 'conductivity quencher', i.e. a density gradient incorporated in the matrix, with the dense region located on the cathodic side (pH 11) for (a) smoothing the voltage gradient on the separation cell and (b) reducing the anodic electrosmotic flow due to the net positive charge acquired by the matrix at pH 11 (1 mM excess protonated amino groups to act as counterions to the 1 mm OH- groups in the bulk water solution generated by the local value of pH 11). Excellent focusing is obtained for such alkaline proteins as lysozyme (pI 10.55), So-6 (a leaf protein, pI 10.49), cytochrome c (pI 10.45) and ribonuclease (pI 10.12).     

Proline uptake by monolayers of human intestinal absorptive (Caco-2) cells in vitro.

Monolayers of the Caco-2 human intestinal cell line exhibit active and passive uptake systems for the imino acid L-proline. The active transport component is saturable and it is responsible for about two thirds of the observed flux over the nanomolar concentration range, at 37 degrees C and pH 7.4. In contrast to L-phenylalanine, specific L-proline uptake has a high degree of sodium dependency and the efficiency of the carrier system is significantly reduced when protein synthesis (cycloheximide), Na+/K(+)-ATPase (ouabain) or cellular metabolism (sodium azide) are inhibited. The expression of the L-proline carrier by Caco-2 cells was under some degree of nutritional control. Glucose deficiency, over the time scale of the experiment, had no effect. The temperature-dependence of the specific uptake process followed the Arrhenius model with an apparent activation energy of 93.5 kJ nmol-1. This pathway also displayed Michaelis-Menten concentration-dependence with a Ksdm of 5.28 mM and a maximal transport flux (Jsdmax) of 835 pmol min-1 (10(6) cells)-1. Although the passive component was unchanged, the pH of the donor phase exerted a profound effect on the active carrier component. Within the physiological pH range a local maximum efficiency was found at pH 7.4 but dramatic increases were noted as pH 5.0 was approached. In competition studies, with 100-fold excess of a second amino acid, strong inhibition of uptake was found with alpha-aminoisobutyric acid, L-alanine and L-serine whereas moderate inhibition was observed with glycine, D-proline and gamma-aminoisobutyric acid. Aromatic and branched amino acids showed weak (L-valine) or no interaction (L-phenylalanine, L-leucine) with the carrier system. These data indicate that the carrier system for the uptake of L-proline has many features in common with the A system for amino acid transport.     

Choroid epithelial cell pH.

The aim of the study was to obtain a reliable estimate of the pH of the parenchymal ($\text{i.e.}$, epithelial) cell compartment of the choroid plexus. From the analysis of the distribution of ¹⁴C-dimethyloxazol-idinedione (DMO) and ³H-inulin in the lateral ventricular plexus, a value of 7.0 can be calculated for choroid cell pH. Because of the presence in choroid plexus of carrier-transport systems for various organic acids, the effect of carrier DMO on the measurement of cell pH was ascertained. The addition of carrier DMO (15 mg/kg, i.p.) did not significantly affect the value of choroid cell pH as calculated from the steady-state distribution of ¹⁴C-DMO. Thus if DMO is actively transported by the choroid plexus, it is probably translocated by a system that is saturated by low concentrations of carrier (ca 1 mg/kg, i.p.).     

Role of spermidine in the activity of sn-glycerol-3-phosphate acyltransferase from Escherichia coli

The integral membrane protein, sn-glycerol-3-phosphate acyltransferase, catalyzes the first committed step in phospholipid synthesis, and both acyl-CoA and acyl-acyl carrier protein can be used as acyl donors in this reaction. We found that spermidine increased the specific activity of the acyltransferase when either substrate was used as the acyl donor. Magnesium, as well as other cations, also increased acyltransferase activity but were not nearly as effective as spermidine. Two roles for spermidine in this reaction were deduced from our data. First, spermidine dramatically lowered the K_m for glycerol 3-phosphate resulting in an overall rate enhancement when either substrate was used as the acyl donor. This effect was attributed to the modification of the acyl-transferase environment due to the binding of spermidine to membrane phospholipids. A second effect of spermidine was evident only when acyl-acyl carrier protein was used as substrate. Using this acyl donor, a pH optimum of 7.5 was found in the absence of spermidine, but in its presence, the pH optimum was shifted to 8.5. Between pH 7.5 and 8.5, palmitoyl-acyl carrier protein undergoes a conformational change to a more expanded, denatured state and its activity in the acyltransferase assay decreases dramatically. Spermidine restored the native conformation of palmitoyl-acyl carrier protein at pH 8.5, thus accounting for the majority of rate enhancement observed at elevated pH.     

Enhanced recombinant protein production and differential expression of molecular chaperones in sf-caspase-1-repressed stable cells after baculovirus infection

Background

Industrial-scale biocatalytic synthesis of fine chemicals occurs preferentially as continuous processes employing immobilized enzymes on insoluble porous carriers. Diffusional effects in these systems often create substrate and product concentration gradients between bulk liquid and the carrier. Moreover, some widely-used biotransformation processes induce changes in proton concentration. Unlike the bulk pH, which is usually controlled at a suitable value, the intraparticle pH of immobilized enzymes may deviate significantly from its activity and stability optima. The magnitude of the resulting pH gradient depends on the ratio of characteristic times for enzymatic reaction and on mass transfer (the latter is strongly influenced by geometrical features of the porous carrier). Design and selection of optimally performing enzyme immobilizates would therefore benefit largely from experimental studies of the intraparticle pH environment. Here, a simple and non-invasive method based on dual-lifetime referencing (DLR) for pH determination in immobilized enzymes is introduced. The technique is applicable to other systems in which particles are kept in suspension by agitation.

Results

The DLR method employs fluorescein as pH-sensitive luminophore and Ru(II) tris(4,7-diphenyl-1,10-phenantroline), abbreviated Ru(dpp), as the reference luminophore. Luminescence intensities of the two luminophores are converted into an overall phase shift suitable for pH determination in the range 5.0-8.0. Sepabeads EC-EP were labeled by physically incorporating lipophilic variants of the two luminophores into their polymeric matrix. These beads were employed as carriers for immobilization of cephalosporin C amidase (a model enzyme of industrial relevance). The luminophores did not interfere with the enzyme immobilization characteristics. Analytical intraparticle pH determination was optimized for sensitivity, reproducibility and signal stability under conditions of continuous measurement. During hydrolysis of cephalosporin C by the immobilizate in a stirred reactor with bulk pH maintained at 8.0, the intraparticle pH dropped initially by about 1 pH unit and gradually returned to the bulk pH, reflecting the depletion of substrate from solution. These results support measurement of intraparticle pH as a potential analytical processing tool for proton-forming/consuming biotransformations catalyzed by carrier-bound immobilized enzymes.

Conclusions

Fluorescein and Ru(dpp) constitute a useful pair of luminophores in by DLR-based intraparticle pH monitoring. The pH range accessible by the chosen DLR system overlaps favorably with the pH ranges at which enzymes are optimally active and stable. DLR removes the restriction of working with static immobilized enzyme particles, enabling suspensions of particles to be characterized also. The pH gradient developed between particle and bulk liquid during reaction steady state is an important carrier selection parameter for enzyme immobilization and optimization of biocatalytic conversion processes. Determination of this parameter was rendered possible by the presented DLR method.     

pH对克雷伯氏菌1,3-丙二醇合成关键酶酶活及发酵特性的影响

在5 L发酵罐进行甘油脉冲流加发酵,分析了不同pH值对克雷伯氏肺炎杆菌发酵特性的影响,pH 6.5为菌体最佳生长条件,克雷伯氏肺炎杆菌合成1,3-丙二醇的产量最高。在1,3-丙二醇合成速率较大的对数中前期,进行甘油脉冲流加发酵,提高甘油浓度促进甘油脱水酶、1,3-丙二醇氧化还原酶和甘油脱氢酶活性。不同pH值的脉冲试验表明,甘油脱水酶,2,3-丁二醇脱氢酶比酶活随着pH值的升高而升高,1,3-丙二醇氧化还原酶,乳酸脱氢酶比酶活在pH6.5最高,因此偏酸性的发酵条件和对数期维持一定的甘油浓度能够促进1,3-丙二醇的合成。     

Potential generation in bilayer lipid membranes in the NADH-flavin mononucleotide-ubiquinone-6-O2 system

Membrane conductance and generation of transmembrane potential by the NADH oxidation reactions in the NADH-flavin mononucleotide-ubiquinone-6-O₂ system have been studied. It has been shown that in solutions of a relatively low buffer capacity at pH 5.8 in the presence of a proton carrier, a potential is generated, the value of which depends on the concentration of the reducer and amounts to 40–60 mV. In the absence of a proton carrier at pH 8, a potential arises, which suggests a transmembrane negative charge transfer. Bilayer lipid membranes have been shown to possess proton selectivity if the reaction is run at pH 3.7. At a pH higher than 5.8 the proton selectivity disappears. Schemes of potential generation in lipid bilayers in different conditions are suggested and discussed.     

Regulation of catalase synthesis in Salmonella typhimurium.

The specific activity of catalase in Salmonella typhimurium and other enteric bacteria decreased during the logarithmic phase of growth and increased at the onset and during the stationary phase. The increase in catalase synthesis at the end of the exponential phase in S. typhimurium cells coincided with the lowest pH value reached by the culture. Maintenance of the pH at a constant neutral value did not alter the typical pattern of synthesis in contradiction of the results previously reported (McCarthy and Hinshelwood. 1959). A sudden decrease in the pH value of an S. typhimurium culture during exponential growth by addition of HC1 did not cause an alteration in the catalase synthesis pattern. Addition of hydrogen peroxide to S. typhimurium cultures within the range 1 muM TO 2MM during the exponential growth phase stimulated catalase synthesis. The extent of catalase synthesis depended on the concentration of hydrogen peroxide; the maximum stimulation was observed at 80 muM. Increased catalase synthesis was not detected for 10 to 15 min after hydrogen peroxide addition. Hydrogen peroxide was produced by S. typhimurium cultures during the exponential and stationary growth phases. However, no direct relationship between hydrogen peroxide accumulation and synthesis of catalase was observed.     

Effect of a pH change in the medium and cytoplasm of cultured cells on the rhythm of protein synthesis

A circahoral rhythm of protein syntheses similar to that in monolayer hepatocytes was discovered in cell culture of Chinese hamster fibroblasts. Studies on the effects of pH changes in the culture medium and cultured cells on different parameters of protein synthesis showed some pH-dependent changes of predecessor pool and of its incorporation intensity into proteins. At the same time changes in a relative incorporation of the predecessor into proteins (with a correction for the pool) were insignificant. This value characterizing the productivity of protein synthesis does not seem to be directly associated with pH changes in the cells. The mean period of the rhythm of protein synthesis and intracellular pH was not changed with medium pH alterations.     

Kinetic and chemical mechanism of the dihydrofolate reductase from Mycobacterium tuberculosis

Dihydrofolate reductase from Mycobacterium tuberculosis (MtDHFR) catalyzes the NAD(P)-dependent reduction of dihydrofolate, yielding NAD(P)(+) and tetrahydrofolate, the primary one-carbon unit carrier in biology. Tetrahydrofolate needs to be recycled so that reactions involved in dTMP synthesis and purine metabolism are maintained. In this work, we report the kinetic characterization of the MtDHFR. This enzyme has a sequential steady-state random kinetic mechanism, probably with a preferred pathway with NADPH binding first. A pK(a) value for an enzymic acid of approximately 7.0 was identified from the pH dependence of V, and the analysis of the primary kinetic isotope effects revealed that the hydride transfer step is at least partly rate-limiting throughout the pH range analyzed. Additionally, solvent and multiple kinetic isotope effects were determined and analyzed, and equilibrium isotope effects were measured on the equilibrium constant. (D(2)O)V and (D(2)O)V/K([4R-4-(2)H]NADH) were slightly inverse at pH 6.0, and inverse values for (D(2)O)V([4R-4-(2)H]NADH) and (D(2)O)V/K([4R-4-(2)H]NADH) suggested that a pre-equilibrium protonation is occurring before the hydride transfer step, indicating a stepwise mechanism for proton and hydride transfer. The same value was obtained for (D)k(H) at pH 5.5 and 7.5, reaffirming the rate-limiting nature of the hydride transfer step. A chemical mechanism is proposed on the basis of the results obtained here.     

Thermodynamic and kinetic control of ATP synthesis in yeast mitochondria: role of delta pH

ATP synthesis rate, measured as the variation in external Pi concentration, varied as a linear function of either delta mu H+ or delta Gpin, in such a manner that the delta Gpin/delta mu H+ ratio increased while VATP increased. We also observed a linear dependence of the flux control coefficient of the Pi carrier on delta pH. All the results presented can be explained by a relatively large delta pH drop when VATP increases.     

Relationship between succinate transport and production of extracellular poly(3-hydroxybutyrate) depolymerase in Pseudomonas lemoignei

The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6. 8. The apparent KS0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [14C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pKa2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate2- accounts for the carbon starvation of P. lemoignei observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products.     

Reactivity of the sulphydryl groups of the mitochondrial phosphate carrier

The rate of reaction of - SH groups of the mitochondrial phosphate carrier with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) and N-ethylmaleimide (MalNEt) was followed by measuring the inhibition of phosphate transport. The changes in the rate of reaction caused by alterations of the ionic composition of the matrix were compared with changes of the total intramitochondrial phosphate content, the intramitochondrial K+ content and the value of intramitochondrial pH. The ionic composition was manipulated by addition of valinomycin to non-respiring or to respiring mitochondria and by addition of inorganic phosphate to respiring and non-respiring mitochondria. From all these variables it was the changes of the intramitochondrial pH which correlated with the - SH group reactivity. Internal acidification decreased and internal alkalinization increased the rate of reaction of mitochondrial phosphate carrier with both Nbs2 and MalNEt. Nbs2 did not penetrate the inner mitochondrial membrane as assayed by determination of the acid-soluble thiol content of the matrix. From this fact it follows that the Nbs2-reactive SH groups of the carrier were accessible from the outer surface of the inner membrane in our experiments. It is concluded that intramitochondrial pH modifies the reactivity of the externally oriented - SH groups indirectly. A hypothesis is presented according to which protonation and deprotonation of the carrier molecule on the inner side could induce a conformational change of the whole protein altering also the microenvironment of the - SH groups near the opposite surface.     

Electrogenic proton transport across lipid bilayer membranes mediated by cationic derivatives of rhodamine 19: comparison with anionic protonophores

Protonophores can be considered as candidates for anti-obesity drugs and tools to prevent excessive reactive oxygen species production in mitochondria by means of a limited decrease in the mitochondrial potential. Experimentally used protonophores are weak acids that can carry protons across a membrane in a neutral (protonated) form, and they come back in an anionic (deprotonated) form. A cationic derivative of rhodamine 19 and plastoquinone (SkQR1) was recently shown to possess uncoupling activity in mitochondria and in intact cells. In this article, we studied the mechanism of action of SkQR1 and its plastoquinone-lacking analog (C₁₂R1) on a planar bilayer lipid membrane by applying voltage jumps. The steady-state current was proportional to the C₁₂R1 concentration in a manner as if the monomeric form of the carrier were operative. As predicted by the carrier model, at high pH, when rhodamines were mainly deprotonated, the current changed immediately following a jump in the applied potential and then remained constant. By contrast, at low pH, the current relaxed from an initially high value to a lower value since the protonated carrier cations were redistributed in the membrane. An inverse pH dependence was revealed with the anionic protonophore CCCP. The dependence of the SkQR1 protonophorous activity on voltage exhibited an increase at high voltages, an effect that might facilitate mild (self-limited) uncoupling of mitochondria.     

Partial purification and characterization of two forms of malonyl-coenzyme A:acyl carrier protein transacylase from soybean leaf tissue

Investigation of malonyl-CoA:acyl carrier protein transacylase from soybeans has shown that this fatty acid biosynthetic enzyme occurs in at least two isozymic forms. Both forms exist as soluble, low-molecular-mass polypeptides (approx 43 kDa) which catalyze one of the first committed steps in the synthesis of C16 and C18 fatty acids. We have partially purified the two forms of this enzyme from soybean leaf tissue 1200- and 3900-fold respectively. Isozyme 1 does not adhere to ion-exchange or blue dye affinity chromatographic supports and elutes from a polybuffer exchanger column at a pH of 7.3. Isozyme 2 requires salt to be eluted from ion-exchange and affinity matrices and elutes from a polybuffer exchanger column at a pH of 5.3. The two forms of malonyl-CoA:acyl carrier protein transacylase also differ in their sensitivity to catalytic inhibitors, heat treatment, and inhibition by acyl-CoA ester substrates. Both forms utilize malonyl-CoA as the preferred substrate, and polyacrylamide gel electrophoresis of reaction products indicated that malonyl-acyl carrier protein was the major product formed. Analysis of developing soybean seeds indicates that only one form (isozyme 1) is predominant, whereas leaf tissue possesses both isozymes.     

Lactose transport system of Streptococcus thermophilus. Functional reconstitution of the protein and characterization of the kinetic mechanism of transport.

The kinetic mechanism of the lactose transport system of Streptococcus thermophilus was studied in membrane vesicles fused with cytochrome c oxidase containing liposomes and in proteoliposomes in which cytochrome c oxidase was coreconstituted with the lactose transport protein. Selective manipulation of the components of the proton (and sodium) motive force indicated that both a membrane potential and a pH gradient could drive transport. The galactoside/proton stoichiometry was close to unity. Experiments which discriminate between the effects of internal pH and delta pH as driving force on galactoside/proton symport showed that the carrier is highly activated at alkaline internal pH values, which biases the transport system kinetically toward the pH component of the proton motive force. Galactoside efflux increased with increasing pH with a pKa of about 8, whereas galactoside exchange (and counterflow) exhibited a pH optimum around 7 with pKa values of 6 and 8, respectively. Imposition of delta pH (interior alkaline) retarded the rate of efflux at any pH value tested, whereas the rate of exchange was stimulated by an imposed delta pH at pH 5.8, not affected at pH 7.0, and inhibited at pH 8.0 and 9.0. The results have been evaluated in terms of random and ordered association/dissociation of galactoside and proton on the inner surface of the membrane. Imposition of delta psi (interior negative) decreased the rate of efflux but had no effect on the rate of exchange, indicating that the unloaded transport protein carries a net negative charge and that during exchange and counterflow the carrier recycles in the protonated form.     

Mechanism of enhanced melibiose transport rate catalyzed by an Escherichia coli lactose carrier mutant with leucine substituted for serine-306. The pH-dependence of melibiose efflux

The mechanism of melibiose transport was studied in cells containing plasmid pAA22 which expresses the mutant lactose carrier (serine-306 to leucine) cloned from Escherichia coli AA22. These studies were of interest because several lines of evidence suggested that the AA22 mutation conferred novel properties upon the lactose carrier, decreasing turnover with several beta-galactoside substrates, increasing turnover with melibiose, and abolishing active accumulation even though equilibration occurred via symport with H+. Although severely defective in active melibiose accumulation, the present study indicates that in cells poisoned with azide the AA22 carrier does in fact equilibrate melibiose across the membrane more rapidly than the normal lactose carrier. Similarly, melibiose efflux from cells preloaded with melibiose was more rapidly catalyzed by the AA22 carrier than by the normal carrier (pH 7.0). Furthermore, although external H+ did reduce net melibiose efflux to a rate slower than seen in equilibrium exchange, a lower than normal pH was required to achieve this effect. Therefore, at pH 7.0, the AA22 carrier (but not the normal carrier) catalyzed net efflux at a rate approaching that for the exchange process (which was pH-resistant in both the mutant and the parent). At pH 8.0 both the AA22 carrier and the normal carrier catalyzed net melibiose efflux at a rate identical to the equilibrium exchange rate. We suggest (i) that the sensitivity of melibiose efflux to external pH indicates that during efflux the AA22 carrier interacts with protons in a manner similar to the normal carrier (i.e., sugar is cotransported with H+) and hence the absence of accumulation is not explained by internal leak via a binary carrier-melibose complex; and (ii) that the modest increase in rate constants for melibiose exit reflect small changes in activation energy (1 kcal/mol) consistent with a steric mechanism possibly involving van der Waals contacts.