Substrate specificity of the oncoprotein v-Fps: site-specific mutagenesis of the putative P+1 pocket

Based on the X-ray structure of the insulin receptor kinase [Hubbard, S. R. (1997) EMBO J. 16, 5572-5581], Arg-1130 in the oncoprotein v-Fps, a nonreceptor tyrosine protein kinase, is predicted to interact with the P+1 glutamate in substrate peptides. To determine whether this residue is an important recognition element in v-Fps, Arg-1130 was substituted with leucine (R1130L) and glutamic acid (R1130E). The ability of these mutants to phosphorylate the peptide EAEIYXAIE, where X is glutamic acid, alanine, or lysine, was assessed. A comparison of the rates of peptide phosphorylation under limiting substrate concentrations (i.e., k(cat)/K(m) conditions) indicates that substrate specificity is altered by the electrostatic environment of the P+1 pocket. When the pocket displays a positive charge (Arg-1130; wild type), no charge (R1130L), or a negative charge (R1130E), v-Fps prefers to phosphorylate the glutamate peptide over the lysine peptide by a 200:1, 9:1, or 1:1 margin. While k(cat)/K(m) for the glutamate peptide is 50-fold higher for wild type compared to R1130E, k(cat)/K(m) for the lysine peptide is 3-fold higher for R1130E compared to wild type, a 150-fold change in relative substrate specificity. Analysis of the individual steps in the kinetic mechanism using viscosometric techniques indicates that the wild-type enzyme binds the glutamate peptide 3-fold better than the alanine peptide and, at least, 10-fold better than the lysine peptide. For R1130L, this margin range is reduced substantially, and for R1130E, no binding preference is observed. Nonetheless, the lysine peptide binds, at least, 4-fold better to R1130E than to wild type, and the glutamate peptide binds 3-fold poorer to R1130E than to wild type. The mutants lower the phosphoryl transfer rate by 4-30-fold for the three peptides, suggesting that Arg-1130 helps to position the tyrosine for optimum catalysis. The data indicate that a single mutation in v-Fps can alter significantly the relative substrate specificity by about 2 orders of magnitude with, at least, 50% of this effect occurring through relative changes in peptide binding affinity.     

Functional significance of a highly conserved glutamate residue of the human noradrenaline transporter

The aim of the study was to investigate the role of glutamate residue 113 in transmembrane domain 2 of the human noradrenaline transporter in determining cell surface expression and functional activity. This residue is absolutely conserved in all members of the Na+- and Cl--dependent transporter family. Mutations to alanine (hE113A), aspartate (hE113D) and glutamine (hE113Q) were achieved by site-directed mutagenesis and the mutants were expressed in transfected COS-7 or HEK-293 cells. Cell surface expression of hE113A and hE113D, but not hE113Q, was markedly reduced compared with wild type, and functional noradrenaline uptake was detected only for the hE113Q mutant. The pharmacological properties of the hE113Q mutant showed very little change compared with wild type, except for a decrease in Vmax values for noradrenaline and dopamine uptake of 2-3-fold. However, the hE113D mutant showed very marked changes in its properties, compared with wild type, with 82-260-fold decreases in the affinities of the substrates, noradrenaline, dopamine and MPP+, and increased Na+ affinity for stimulation of nisoxetine binding. The results of the study show that the size and not the charge of the 113 glutamate residue of the noradrenaline transporter seems to be the most critical factor for maintenance of transporter function and surface expression.     

Aspartate 345 of the dopamine transporter is critical for conformational changes in substrate translocation and cocaine binding

The present study elucidated the role of aspartate 345, a residue conserved in the third intracellular loop of all Na+/Cl(-)-dependent neurotransmitter transporters, in conformational changes of the dopamine (DA) transporter. Asparagine substitution (D345N) resulted in near normal transporter expression on the cell surface but caused extremely low Vmax and Km values for DA uptake, converted the inhibitory effect of Zn2+ on DA uptake to a stimulatory one, and eliminated reverse transport. The cocaine-like inhibitor 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane or the selective DA transporter inhibitor GBR12935 bound to D345N with a normal affinity and still inhibited DA uptake potently. However, the mutation reduced the binding capacity of the surface transporter for these two inhibitors by 90% or more. Moreover, the binding activity of D345N can be significantly improved by Zn2+ but not by Na+. These results are consistent with a defect in reorientation of the substrate-binding site to the extracellular side, leading to a loss of the outward-facing conformational state where external DA binds to initiate uptake and the inhibitors bind to initiate uptake inhibition. Alanine or glutamate substitution produced a similar phenotype, suggesting that both the negative charge and the residue volume at position 345 are vital. Furthermore, in intact cells, cocaine potentiated the reaction of the membrane-impermeant sulfhydryl reagent methanethiosulfonate ethyltrimethylammonium with the extracellularly located endogenous cysteines of D345N but not those of wild type, and this potentiation was blocked upon K+ substitution for Na+. Thus, cocaine binding to D345N likely induces a different and Na(+)-dependent conformational change, which may contribute to its Na(+)-dependent uptake inhibitory activity.     

Site-directed mutation of arginine 282 to glutamate uncouples the movement of peptides and protons by the rabbit proton-peptide cotransporter PepT1

A conserved positive residue in the seventh transmembrane domain of the mammalian proton-coupled di- and tripeptide transporter PepT1 has been shown by site-directed mutagenesis to be a key residue for protein function. Substitution of arginine 282 with a glutamate residue (R282E-PepT1) gave a protein at the plasma membrane of Xenopus laevis oocytes that was able to transport the non-hydrolyzable dipeptide [3H]d-Phe-l-Gln, although unlike the wild type, the rate of transport by R282E-PepT1 was independent of the extracellular pH level, and the substrate could not be accumulated above equilibrium. The binding affinity of the mutant transport protein was unchanged from the wild type. Thus, R282E-Pept1 appears to have been changed from a proton-driven to a facilitated transporter for peptides. In addition, peptide transport by R282E-PepT1 still induced depolarization as measured by microelectrode recordings of membrane potential. A more detailed study by two-electrode voltage clamping revealed that R282E-PepT1 behaved as a peptide-gated non-selective cation channel with the ion selectivity series lithium > sodium > N-methyl-d-glucamine at pH 7.4. There was also a proton conductance (comparing pH 7.4 and 8.4), and at pH 5.5 the predominant conductance was for potassium ions. Therefore, it can be concluded that changing arginine 282 to a glutamate not only uncouples the cotransport of protons and peptides of the wild-type PepT1 but also creates a peptide-gated cation channel in the protein.     

Slow photo-cross-linking kinetics of benzophenone-labeled voltage sensors of ion channels

Voltage-gated ion channels have voltage sensors that move in response to changes in membrane potential. This movement regulates the gates that control access of ions to the permeation pathway. To study the coupling between voltage sensors and gates, we immobilize the voltage sensors, using a bifunctional photo-cross-linking reagent that can be attached to an introduced cysteine, and observe the consequences for gate movement [Horn, R., Ding, S., and Gruber, H. J. (2000) J. Gen. Physiol. 116, 461-475]. UV irradiation of the benzophenone adduct attached to the cysteine residue immobilizes the voltage sensors, S4 segments, of both Na(+) and Shaker K(+) channels. Here we examine the kinetics of S4 immobilization after a brief UV flash. Immobilization has an exponential time course with time constants of >200 ms for Shaker and 17 ms for Na(+) channels, whereas the triplet excited state lifetime of the benzophenone adduct is <1 ms. This result suggests that H-atom abstraction by benzophenone is rapid and that the rate-limiting step in immobilization is the recombination of alkyl and ketyl free radicals generated by H-abstraction. H-Abstraction is also 2.7-fold more efficient at a hyperpolarized voltage than at a depolarized membrane potential in Shaker S4 segments. S4 immobilization after a UV flash can be prevented by depolarization of Shaker channels, suggesting that movement in the activation pathway is capable of separating the ketyl and alkyl free radicals. Exploiting the unique charge movement and gating properties of the L382V mutant of Shaker, we show that free radical separation follows S4 movement itself and is relatively independent of the movement of activation gates.     

Glutamate 268 regulates transport probability of the anion/proton exchanger ClC-5

The Cl(-)/H(+) exchange mediated by ClC transporters can be uncoupled by external SCN(-) and mutations of the proton glutamate, a conserved residue at the internal side of the protein. We show here for the mammalian ClC transporter ClC-5 that acidic internal pH led to a greater increase in currents upon exchanging extracellular Cl(-) for SCN(-). However, transport uncoupling, unitary current amplitudes, and the voltage dependence of the depolarization-induced activation were not altered by low pH values. Therefore, it is likely that an additional gating process regulates ClC-5 transport. Higher internal [H(+)] and the proton glutamate mutant E268H altered the ratio between ClC-5 transport and nonlinear capacitance, indicating that the gating charge movements in ClC-5 arise from incomplete transport cycles and that internal protons increase the transport probability of ClC-5. This was substantiated by site-directed sulfhydryl modification of the proton glutamate mutant E268C. The mutation exhibited small transport currents together with prominent gating charge movements. The charge restoration using a negatively charged sulfhydryl reagent reinstated also the WT phenotype. Neutralization of the charge of the gating glutamate 211 by the E211C mutation abolished the effect of internal protons, showing that the increased transport probability of ClC-5 results from protonation of this residue. S168P (a mutation that decreases the anion affinity of the central binding site) reduced also the internal pH dependence of ClC-5. These results support the idea that protonation of the gating glutamate 211 at the central anion-binding site of ClC-5 is mediated by the proton glutamate 268.     

A single carboxyl mutant of the multidrug transporter EmrE is fully functional

EmrE, a multidrug transporter from Escherichia coli removes toxic compounds from the cell in exchange with protons. Glu-14 is the only charged residue in the putative membrane domains and is fully conserved in more than 50 homologues of the protein. This residue was shown to be an essential part of the binding site, common to protons and substrate. EmrE bearing a single carboxylic residue, Glu-14, shows uptake and binding properties similar to those of the wild type. This suggests that a small protein bearing only 110 amino acids with a single carboxyl in position 14 is the most basic structure that shows ion-coupled transport activity. The role of Glu-14 in substrate binding was examined by using dicyclohexylcarbodiimide, a hydrophobic carbodiimide that is known to react with carboxyls. Tetraphenylphosphonium binding to both wild type and the single carboxyl mutant is inhibited by dicyclohexylcarbodiimide in a dose-dependent manner. Ethidium and other substrates of EmrE prevent this inhibition with an order of potency in accord with their apparent affinities. This suggests that dicyclohexylcarbodiimide binding is sterically prevented by the substrate, supporting the contention that Glu-14, the reactive residue, is part of the substrate-binding site.     

Conserved asparagine residue located in binding pocket controls cation selectivity and substrate interactions in neuronal glutamate transporter

Transporters of the major excitatory neurotransmitter glutamate play a crucial role in glutamatergic neurotransmission by removing their substrate from the synaptic cleft. The transport mechanism involves co-transport of glutamic acid with three Na(+) ions followed by countertransport of one K(+) ion. Structural work on the archeal homologue Glt(Ph) indicates a role of a conserved asparagine in substrate binding. According to a recent proposal, this residue may also participate in a novel Na(+) binding site. In this study, we characterize mutants of this residue from the neuronal transporter EAAC1, Asn-451. None of the mutants, except for N451S, were able to exhibit transport. However, the K(m) of this mutant for l-aspartate was increased ～30-fold. Remarkably, the increase for d-aspartate and l-glutamate was 250- and 400-fold, respectively. Moreover, the cation specificity of N451S was altered because sodium but not lithium could support transport. A similar change in cation specificity was observed with a mutant of a conserved threonine residue, T370S, also implicated to participate in the novel Na(+) site together with the bound substrate. In further contrast to the wild type transporter, only l-aspartate was able to activate the uncoupled anion conductance by N451S, but with an almost 1000-fold reduction in apparent affinity. Our results not only provide experimental support for the Na(+) site but also suggest a distinct orientation of the substrate in the binding pocket during the activation of the anion conductance.     

Functional consequences of phosphomimetic mutations at key cAMP-dependent protein kinase phosphorylation sites in the type 1 inositol 1,4,5-trisphosphate receptor

Regulation of Ca(2+) release through inositol 1,4,5-trisphosphate receptors (InsP(3)R) has important consequences for defining the particular spatio-temporal properties of intracellular Ca(2+) signals. In this study, regulation of Ca(2+) release by phosphorylation of type 1 InsP(3)R (InsP(3)R-1) was investigated by constructing "phosphomimetic" charge mutations in the functionally important phosphorylation sites of both the S2+ and S2- InsP(3)R-1 splice variants. Ca(2+) release was investigated following expression in Dt-40 3ko cells devoid of endogenous InsP(3)R. In cells expressing either the S1755E S2+ or S1589E/S1755E S2- InsP(3)R-1, InsP(3)-induced Ca(2+) release was markedly enhanced compared with nonphosphorylatable S2+ S1755A and S2- S1589A/S1755A mutants. Ca(2+) release through the S2- S1589E/S1755E InsP(3)R-1 was enhanced approximately 8-fold over wild type and approximately 50-fold when compared with the nonphosphorylatable S2- S1589A/S1755A mutant. In cells expressing S2- InsP(3)R-1 with single mutations in either S1589E or S1755E, the sensitivity of Ca(2+) release was enhanced approximately 3-fold; sensitivity was midway between the wild type and the double glutamate mutation. Paradoxically, forskolin treatment of cells expressing either single Ser/Glu mutation failed to further enhance Ca(2+) release. The sensitivity of Ca(2+) release in cells expressing S2+ S1755E InsP(3)R-1 was comparable with the sensitivity of S2- S1589E/S1755E InsP(3)R-1. In contrast, mutation of S2+ S1589E InsP(3)R-1 resulted in a receptor with comparable sensitivity to wild type cells. Expression of S2- S1589E/S1755E InsP(3)R-1 resulted in robust Ca(2+) oscillations when cells were stimulated with concentrations of alpha-IgM antibody that were threshold for stimulation in S2- wild type InsP(3)R-1-expressing cells. However, at higher concentrations of alpha-IgM antibody, Ca(2+) oscillations of a similar period and magnitude were initiated in cells expressing either wild type or S2- phosphomimetic mutations. Thus, regulation by phosphorylation of the functional sensitivity of InsP(3)R-1 appears to define the threshold at which oscillations are initiated but not the frequency or amplitude of the signal when established.     

Identification of a novel Na+-independent acidic amino acid transporter with structural similarity to the member of a heterodimeric amino acid transporter family associated with unknown heavy chains

We identified a novel Na(+)-independent acidic amino acid transporter designated AGT1 (aspartate/glutamate transporter 1). AGT1 exhibits the highest sequence similarity (48% identity) to the Na(+)-independent small neutral amino acid transporter Asc (asc-type amino acid transporter)-2 a member of the heterodimeric amino acid transporter family presumed to be associated with unknown heavy chains (Chairoungdua, A., Kanai, Y., Matsuo, H., Inatomi, J., Kim, D. K., and Endou, H. (2001) J. Biol. Chem. 276, 49390-49399). The cysteine residue responsible for the disulfide bond formation between transporters (light chains) and heavy chain subunits of the heterodimeric amino acid transporter family is conserved for AGT1. Because AGT1 solely expressed or coexpressed with already known heavy chain 4F2hc (4F2 heavy chain) or rBAT (related to b(0,+)-amino acid transporter) did not induce functional activity, we generated fusion proteins in which AGT1 was connected with 4F2hc or rBAT. The fusion proteins were sorted to the plasma membrane and expressed the Na(+)-independent transport activity for acidic amino acids. Distinct from the Na(+)-independent cystine/glutamate transporter xCT structurally related to AGT1, AGT1 did not accept cystine, homocysteate, and l-alpha-aminoadipate and exhibited high affinity to aspartate as well as glutamate, suggesting that the negative charge recognition site in the side chain-binding site of AGT1 would be closer to the alpha-carbon binding site compared with that of xCT. The AGT1 message was predominantly expressed in kidney. In mouse kidney, AGT1 protein was present in the basolateral membrane of the proximal straight tubules and distal convoluted tubules. In the Western blot analysis, AGT1 was detected as a high molecular mass band in the nonreducing condition, whereas the band shifted to a 40-kDa band corresponding to the AGT1 monomer in the reducing condition, suggesting the association of AGT1 with other protein via a disulfide bond. The finding of AGT1 and Asc-2 has established a new subgroup of the heterodimeric amino acid transporter family whose members associate not with 4F2hc or rBAT but with other unknown heavy chains.     

Reaction mechanism of the reconstituted aspartate/glutamate carrier from bovine heart mitochondria

A functional model for the aspartate/glutamate carrier of the inner mitochondrial membrane was established based on a kinetic evaluation of this transporter. Antiport kinetics were measured in proteoliposomes that contained partially purified carrier protein of definite transmembrane orientation (Dierks, T. and Kr?mer, R. (1988) Biochim. Biophys. Acta 937, 122-126). Bireactant initial velocity analyses of the counterexchange reaction were carried out varying substrate concentrations both in the internal and the external compartment. The kinetic patterns obtained were inconsistent with a pong-pong mechanism; rather they demonstrated the formation of a ternary complex as a consequence of sequential binding of one internal and one external substrate molecule to the carrier. Studies on transport activity in the presence of aspartate and glutamate in the same compartment (formally treated as substrate inhibition) clearly indicated that during exchange only one form of the carrier at either membrane surface exposes its binding sites, for which the two different substrates compete. In the deenergized state (pH 6.5) both substrates were translocated at about the same rate. Aspartate/glutamate antiport became asymmetric if a membrane potential was imposed, due to the electrogenic nature of the heteroexchange resulting from proton cotransport together with glutamate. Investigation of the electrical properties of aspartate/aspartate homoexchange led to the conclusion that the translocating carrier-substrate intermediate exhibits a transmembrane symmetry with respect to the (negative) charge, which again only is conceivable assuming a ternary complex. Thus, an antiport model is outlined that shows the functional complex of the carrier with two substrate molecules bound, one at either side of the membrane. The conformational change associated with the transition of both substrate molecules across the membrane then occurs in a single step. Furthermore the model implicates a distinct proton binding site, which is derived from the different influence of H+ concentration observed on transport affinity and transport velocity, respectively, when glutamate is used as a substrate.     

Histidine scanning mutagenesis of basic residues of the S4 segment of the shaker k+ channel

The voltage sensor of the Shaker potassium channel is comprised mostly of positively charged residues in the putative fourth transmembrane segment, S4 (Aggarwal, S.K., and R. MacKinnon. 1996. Neuron. 16:1169-1177; Seoh, S.-A., D. Sigg, D.M. Papazian, and F. Bezanilla. 1996. Neuron. 16:1159-1167). Movement of the voltage sensor in response to a change in the membrane potential was examined indirectly by measuring how the accessibilities of residues in and around the sensor change with voltage. Each basic residue in the S4 segment was individually replaced with a histidine. If the histidine tag is part of the voltage sensor, then the gating charge displaced by the voltage sensor will include the histidine charge. Accessibility of the histidine to the bulk solution was therefore monitored as pH-dependent changes in the gating currents evoked by membrane potential pulses. Histidine scanning mutagenesis has several advantages over other similar techniques. Since histidine accessibility is detected by labeling with solution protons, very confined local environments can be resolved and labeling introduces minimal interference of voltage sensor motion. After histidine replacement of either residue K374 or R377, there was no titration of the gating currents with internal or external pH, indicating that these residues do not move in the transmembrane electric field or that they are always inaccessible. Histidine replacement of residues R365, R368, and R371, on the other hand, showed that each of these residues traverses entirely from internal exposure at hyperpolarized potentials to external exposure at depolarized potentials. This translocation enables the histidine to transport protons across the membrane in the presence of a pH gradient. In the case of 371H, depolarization drives the histidine to a position that forms a proton pore. Kinetic models of titrateable voltage sensors that account for proton transport and conduction are presented. Finally, the results presented here are incorporated into existing information to propose a model of voltage sensor movement and structure.     

A role for the middle C terminus of G-protein-activated inward rectifier potassium channels in regulating gating

We have used sulfhydryl-modifying reagents to investigate the regulation of G-protein-activated inward rectifier potassium (GIRK) channels via their cytoplasmic domains. Modification of either the conserved N-terminal cysteines (GIRK1C53 and GIRK2C65) or the middle C-terminal cysteines (GIRK1C310 and GIRK2C321) independently inhibited GIRK1/GIRK2 heteromeric channels. With the exception of GIRK2C65, these cysteines were relatively inaccessible to large modifying reagents. The accessibility was further reduced by a mutation at the end of the second transmembrane domain that stabilized the open state of the channel. Thus it is unlikely that these cysteines line the permeation pathway of the open pore. Cysteines introduced 3 and 6 amino acids upstream of GIRK2C321 (G318C and E315C) were considerably more accessible. The effect of modification was dependent on the charge of the reagent. Modification of E315C in GIRK2 and E304C in GIRK1 by sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increased the current by approximately 17-fold, whereas modification by 2-aminoethyl methanethiosulfonate hydrochloride (MTSEA(+)), abolished the current. There was no effect on single-channel conductance. Thus a switch in charge at this middle C-terminal position was sufficient to gate the channel open and closed. This glutamate is conserved in all members of the Kir family. The E303K mutation in Kir2.1 inhibits channel function and causes Andersen's syndrome in humans (Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R., Clark, J., Deymeer, F., George, A. L., Jr., Fish, F. A., Hahn, A., Nitu, A., Ozdemir, C., Serdaroglu, P., Subramony, S. H., Wolfe, G., Fu, Y. H., and Ptacek, L. J. (2001) Cell 105, 511-519 and Preisig-Muller, R., Schlichthorl, G., Goerge, T., Heinen, S., Bruggemann, A., Rajan, S., Derst, C., Veh, R. W., and Daut, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7774-7779). Our results suggest that this residue regulates channel gating through an electrostatic mechanism.     

The molecular basis of glyphosate resistance by an optimized microbial acetyltransferase

GAT is an N-acetyltransferase from Bacillus licheniformis that was optimized by gene shuffling for acetylation of the broad spectrum herbicide, glyphosate, forming the basis of a novel mechanism of glyphosate tolerance in transgenic plants (Castle, L. A., Siehl, D. L., Gorton, R., Patten, P. A., Chen, Y. H., Bertain, S., Cho, H. J., Duck, N., Wong, J., Liu, D., and Lassner, M. W. (2004) Science 304, 1151-1154). The 1.6-A resolution crystal structure of an optimized GAT variant in ternary complex with acetyl coenzyme A and a competitive inhibitor, 3-phosphoglyerate, defines GAT as a member of the GCN5-related family of N-acetyltransferases. Four active site residues (Arg-21, Arg-73, Arg-111, and His-138) contribute to a positively charged substrate-binding site that is conserved throughout the GAT subfamily. Structural and kinetic data suggest that His-138 functions as a catalytic base via substrate-assisted deprotonation of the glyphosate secondary amine, whereas another active site residue, Tyr-118, functions as a general acid. Although the physiological substrate is unknown, native GAT acetylates D-2-amino-3-phosphonopropionic acid with a kcat/Km of 1500 min-1 mM-1. Kinetic data show preferential binding of short analogs to native GAT and progressively better binding of longer analogs to optimized variants. Despite a 200-fold increase in kcat and a 5.4-fold decrease in Km for glyphosate, only 4 of the 21 substitutions present in R7 GAT lie in the active site. Single-site revertants constructed at these positions suggest that glyphosate binding is optimized through substitutions that increase the size of the substrate-binding site. The large improvement in kcat is likely because of the cooperative effects of additional substitutions located distal to the active site.     

Functional Defects in the External and Internal Thin Gates of the γ-Aminobutyric Acid (GABA) Transporter GAT-1 Can Compensate Each Other

The GABA transporter GAT-1 belongs to the neurotransmitter:sodium:symporters which are crucial for synaptic transmission. GAT-1 mediates electrogenic transport of GABA together with sodium and chloride. Structure-function studies indicate that the bacterial homologue LeuT, which possess extra- and intracellular thin gates, is an excellent model for this class of neurotransmitter transporters. We recently showed that a conserved aspartate residue of GAT-1, Asp-451, whose LeuT equivalent participates in its thin extracellular gate, is functionally irreplaceable in GAT-1. Only the D451E mutant exhibited residual transport activity but with an elevated apparent sodium affinity as a consequence of an increased proportion of outward-facing transporters. Because during transport the opening and closing of external and internal gates should be tightly coupled, we have addressed the question of whether mutations of the intracellular thin gate residues Arg-44 and Asp-410 can compensate for the effects of their extracellular counterparts. Mutation of Asp-410 to glutamate resulted in impaired transport activity and a reduced apparent affinity for sodium. However, the transport activity of the double mutant D410E/D451E was increased by approximately 10-fold of that of each of the single mutants. Similar compensatory effects were also seen when other combinations of intra- and extracellular thin gate mutants were analyzed. Moreover, the introduction of D410E into the D451E background resulted in lower apparent sodium affinity than that of D451E alone. Our results indicate that a functional interaction of the external and internal gates of GAT-1 is essential for transport.     

Labeling of specific lysine residues at the active site of glutamine synthetase

Glutamine synthetase (Escherichia coli) was incubated with three different reagents that react with lysine residues, viz. pyridoxal phosphate, 5'-p-fluorosulfonylbenzoyladenosine, and thiourea dioxide. The latter reagent reacts with the epsilon-nitrogen of lysine to produce homoarginine as shown by amino acid analysis, nmr, and mass spectral analysis of the products. A variety of differential labeling experiments were conducted with the above three reagents to label specific lysine residues. Thus pyridoxal phosphate was found to modify 2 lysine residues leading to an alteration of catalytic activity. At least 1 lysine residue has been reported previously to be modified by pyridoxal phosphate at the active site of glutamine synthetase (Whitley, E. J., and Ginsburg, A. (1978) J. Biol. Chem. 253, 7017-7025). By varying the pH and buffer, one or both residues could be modified. One of these lysine residues was associated with approximately 81% loss in activity after modification while modification of the second lysine residue led to complete inactivation of the enzyme. This second lysine was found to be the residue which reacted specifically with the ATP affinity label 5'-p-fluorosulfonylbenzoyladenosine. Lys-47 has been previously identified as the residue that reacts with this reagent (Pinkofsky, H. B., Ginsburg, A., Reardon, I., Heinrikson, R. L. (1984) J. Biol. Chem. 259, 9616-9622; Foster, W. B., Griffith, M. J., and Kingdon, H. S. (1981) J. Biol. Chem. 256, 882-886). Thiourea dioxide inactivated glutamine synthetase with total loss of activity and concomitant modification of a single lysine residue. The modified amino acid was identified as homoarginine by amino acid analysis. The lysine residue modified by thiourea dioxide was established by differential labeling experiments to be the same residue associated with the 81% partial loss of activity upon pyridoxal phosphate inactivation. Inactivation with either thiourea dioxide or pyridoxal phosphate did not affect ATP binding but glutamate binding was weakened. The glutamate site was implicated as the site of thiourea dioxide modification based on protection against inactivation by saturating levels of glutamate. Glutamate also protected against pyridoxal phosphate labeling of the lysine consistent with this residue being the common site of reaction with thiourea dioxide and pyridoxal phosphate.     

The protein sequence responsible for lipoprotein membrane localization in Escherichia coli exhibits remarkable specificity

Structural information defining an N-terminal sequence required for the membrane sorting of bacterial lipoproteins has been previously garnered through the study of a hybrid outer membrane (OM) lipo-beta-lactamase (LL) (Ghrayeb and Inouye (1984) J. Biol. Chem. 259, 463-467). Introduction of an aspartate as the second residue of mature LL (D2 mutant) causes an inner membrane (IM) localization of this protein (Yamaguchi, K., Yu, F., and Inouye, M. (1988) Cell 53, 423-432). Introduction of as aspartate at the third residue of mature LL (D3) causes a weaker IM sorting signal and when present as the fourth residue (D4), normal OM sorting occurs. A positively charged residue at the second position (K2) has no effect on OM localization. Remarkably, glutamate substitution at either the second (E2) or third (E3) position does not interfere with OM sorting. Sorting of the mutant D2 LL can be partially suppressed by introduction of a positively charged histidine (D2H3) or lysine (D2K3) at residue 3 of the mature protein. These results indicate that both the negative charge of the aspartate residue and some structural feature not present in a glutamate residue are required for sorting to the IM. The suppression of IM localization of the D2H3 LL double mutant can be eliminated by growing Escherichia coli at pH 8.4 to reduce the histidine partial positive charge. This result supports the essentiality of a negative charge in IM localization and indicates that the committed step in lipoprotein sorting is made in a cellular compartment, the periplasm, at equilibrium with the external pH.     

Study of active site topography of rat liver mitochondrial dicarboxylate transporter using lipophilic substrate derivatives

Earlier it has been demonstrated that the active site (substrate-binding site + active site channel) of rat liver mitochondrial dicarboxylate transporter is characterized by rather complex topography. Probing the active site with 2-monoalkylmalonates revealed the existence of internal and external lipophilic areas separated by a polar region. A two substrate-binding site model of the transporter has been supposed. The correctness of this model has been evaluated by probing the active site with O-acyl-L-malates differing from 2-monoalkylmalonates by 0.23 nm longer distance from the anion groups to the aliphatic chain. Changes in the polar group of the probe did not prevent its binding and showed the same variable lipophilicity pattern for the transporter channel. Probing with alpha,omega-alkylene dimalonates did not reveal the second substrate-binding site at the active site. The substrate-binding site did not show any differences in affinity to O-acyl-derivatives of L-malate and D-malate, except L-malate binds more effectively than D-malate. This suggests involvement of the L-malate hydroxyl group in substrate binding and stereospecific behavior of the transporter substrate-binding site. A modified one substrate-binding site model of the dicarboxylate transporter is discussed.     

Association of catalytic and regulatory subunits of cyclic AMP-dependent protein kinase requires a negatively charged side group at a conserved threonine.

In Saccharomyces cerevisiae, as in higher eucaryotes, cyclic AMP (cAMP)-dependent protein kinase is a tetramer composed of two catalytic (C) subunits and two regulatory (R) subunits. In the absence of cAMP, the phosphotransferase activity of the C subunit is inhibited by the tight association with R. Mutation of Thr-241 to Ala in the C1 subunit of S. cerevisiae reduces the affinity of this subunit for the R subunit approximately 30-fold and results in a monomeric cAMP-independent C subunit. The analogous residue in the mammalian C subunit is known to be phosphorylated. Peptide maps of in vivo 32P-labeled wild-type C1 and mutant C1(Ala241) suggest that Thr-241 is phosphorylated in yeast cells. Substituting Thr-241 with either aspartate or glutamate partially restored affinity for the R subunit. Uncharged and positively charged residues substituted at this site resulted in C subunits that failed to associate with the R subunit. Replacement with the phosphorylatable residue serine resulted in a C subunit with wild-type affinity for the R subunit. Analysis of this protein revealed that it appears to be phosphorylated on Ser-241 in vivo. These data suggest that the interaction between R and C involves a negatively charged phosphothreonine at position 241 of yeast C1, which can be mimicked by either aspartate, glutamate, or phosphoserine.     

Function of arginine-166 in the active site of Escherichia coli alkaline phosphatase

The function of arginine residue 166 in the active site of Escherichia coli alkaline phosphatase was investigated by site-directed mutagenesis. Two mutant versions of alkaline phosphatase, with either serine or alanine in the place of arginine at position 166, were generated by using a specially constructed M13 phage carrying the wild-type phoA gene. The mutant enzymes with serine and alanine at position 166 have very similar kinetic properties. Under conditions of no external phosphate acceptor, the kcat for the mutant enzymes decreases by approximately 30-fold while the Km increases by less than 2-fold. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, the kcat for the mutant enzymes is reduced by less than 3-fold, while the Km increases by more than 50-fold. For both mutant enzymes, in either the absence or the presence of a phosphate acceptor, the catalytic efficiency as measured by the kcat/Km ratio decreases by approximately 50-fold as compared to the wild type. Measurements of the Ki for inorganic phosphate show an increase of approximately 50-fold for both mutants. Phenylglyoxal, which inactivates the wild-type enzyme, does not inactivate the Arg-166----Ala enzyme. This result indicates that Arg-166 is the same arginine residue that when chemically modified causes loss of activity [Daemen, F.J.M., & Riordan, J.F. (1974) Biochemistry 13, 2865-2871]. The data reported here suggest that although Arg-166 is important for activity is not essential. The analysis of the kinetic data also suggests that the loss of arginine-166 at the active site of alkaline phosphatase has two different effects on the enzyme. First, the binding of the substrate, and phosphate as a competitive inhibitor, is reduced; second, the rate of hydrolysis of the covalent phosphoenzyme may be diminished.