Functional analysis of polar amino-acid residues in membrane associated regions of the NHE1 isoform of the mammalian Na+/H+ exchanger.

The NHE1 isoform of the Na+/H+ exchanger is a ubiquitous plasma membrane protein that regulates intracellular pH in mammalian cells. Site-specific mutagenesis was used to examine the functional role of conserved, polar amino-acid residues occurring in segments of the protein associated with the membrane. Seventeen mutant proteins were assessed by characterization of intracellular pH changes in stably transfected cells that lacked an endogenous Na+/H+ exchanger. All of the mutant proteins were targeted correctly to the plasma membrane and were expressed at similar levels. Amino-acid residues Glu262 and Asp267 were critical to Na+/H+ exchanger activity while mutation of Glu391 resulted in only a partial reduction in activity. The Glu262-->Gln mutant was expressed partially as a deglycosylated protein with increased sensitivity to trypsin treatment in presence of Na+. Substitution of mutated Glu262, Asp267 and Glu391 with alternative acidic residues restored Na+/H+ exchanger activity. The Glu262-->Asp mutant had a decreased affinity for Li+, but its activity for Na+ and H+ ions was unaffected. The results support the hypothesis that side-chain oxygen atoms in a few, critically placed amino acids are important in Na+/H+ exchanger activity and the acidic amino-acid residues at positions 262, 267 and 391 are good candidates for being involved in Na+ coordination by the protein.     

Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. The role of a conserved sequence motif, GXXXXRXGRR, in a putative cytoplasmic loop between helices 2 and 3.

The region including the conserved Ser65-Asp66 dipeptide in the tetracycline/H+ antiporter (TET) encoded by transposon Tn10 is thought to play a gating role (Yamaguchi, A., Ono, N., Akasaka, T., Noumi, T., and Sawai, T. (1990) J. Biol. Chem. 265, 15525-15530). The dipeptide is in putative interhelix loop2-3, which also includes the conserved sequence motif, GXXXXRXGRR, found in all TET proteins and sugar/H+ symporters. Through the combination of localized random and site-directed mutagenesis, each residue in loop2-3 was replaced. Among 10 residues in putative loop2-3, the important residues, of which substitution resulted in significant reduction or complete loss of the transport activity, were Gly62, Asp66, Gly69, and Arg70. The defect in the transport activity of the Gly62 and Gly69 substitution mutants corresponded to the steric hindrance by the substituents as to the putative beta-turn structure of the peptide backbone containing these glycines. Of 3 conserved Arg residues, the replacement of only Arg70 caused complete loss of the activity except for replacement with Lys, indicating the importance of a positive charge at this position, which is similar to the essentiality of a negative charge at Asp66. A "charge-neutralizing" intra-loop salt bridge between Asp66 and Arg70 was not likely because the double mutant in which Asp66 and Arg70 were replaced with asparagine and leucine, respectively, showed no transport activity. A triple mutant with only one positive charge at Arg70 in this loop showed about half the wild-type activity, indicating that the polycationic nature of the loop was not critical for the activity. Cys mutants as to the unessential residues in the loop were modifiable with N-ethylmaleimide, except for the Met64----Cys and Arg71----Cys mutants; however, the modification of only the Ser65----Cys mutant caused significant inhibition of the transport activity, indicating that position 65 is a unique position in the structure of loop2-3.     

Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. Roles of the aspartyl residues located in the putative transmembrane helices.

Three conserved aspartyl residues located in the putative transmembrane helices in the Tn10-encoded metal-tetracycline/H+ antiporter were replaced by Asn, Lys, or Glu with oligonucleotide-directed site-specific mutagenesis. Replacement of Asp84 or Asp15 by Asn or Lys caused a severe defect in tetracycline transport activity, however, the Glu84 and Glu15 mutants retained 150 and 40% of the wild type activity, respectively, indicating the critical role of the negative charge. The increase in the activity of the Glu84 mutant was due to an increase in the affinity for the substrate. H+/tetracycline coupling was intact in these mutants, including Asn and Lys mutants. On the other hand, all of the Asp285-substitution mutants showed a severe defect in tetracycline transport activity and a complete lack of tetracycline-coupled H+ transport. However, since in vivo tests showed the tetracycline resistance for the Glu285 mutant, a negative charge in position 285 plays some role in maintaining the possible down-hill and/or low affinity efflux of accumulated tetracycline from intact cells. Similar work was done for Asp365, and here the Asn and Glu mutants showed decreased but high activity, while the Lys mutant was only marginally active (5%), indicating that a negative charge is not so demanding in position 365, possibly because it is not in the membrane.     

Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase

Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase) uses PP(i) as an energy donor and requires free Mg(2+) for enzyme activity and stability. To determine the catalytic domain, we analyzed charged residues (Asp(253), Lys(261), Glu(263), Asp(279), Asp(283), Asp(287), Asp(723), Asp(727), and Asp(731)) in the putative PP(i)-binding site and two conserved acidic regions of mung bean V-PPase by site-directed mutagenesis and heterologous expression in yeast. Amino acid substitution of the residues with alanine and conservative residues resulted in a marked decrease in PP(i) hydrolysis activity and a complete loss of H(+) transport activity. The conformational change of V-PPase induced by the binding of the substrate was reflected in the susceptibility to trypsin. Wild-type V-PPase was completely digested by trypsin but not in the presence of Mg-PP(i), while two V-PPase mutants, K261A and E263A, became sensitive to trypsin even in the presence of the substrate. These results suggest that the second acidic region is also implicated in the substrate hydrolysis and that at least two residues, Lys(261) and Glu(263), are essential for the substrate-binding function. From the observation that the conservative mutants K261R and E263D showed partial activity of PP(i) hydrolysis but no proton pump activity, we estimated that two residues, Lys(261) and Glu(263), might be related to the energy conversion from PP(i) hydrolysis to H(+) transport. The importance of two residues, Asp(253) and Glu(263), in the Mg(2+)-binding function was also suggested from the trypsin susceptibility in the presence of Mg(2+). Furthermore, it was found that the two acidic regions include essential common motifs shared among the P-type ATPases.     

Functional involvement of membrane-embedded and conserved acidic residues in the ShaA subunit of the multigene-encoded Na+/H+ antiporter in Bacillus subtilis

ShaA, a member of a multigene-encoded Na+/H+ antiporter in B. subtilis, is a large integral membrane protein consisting of 20 transmembrane helices (TM). Conservation of ShaA-like protein subunits in several cation-coupled enzymes, including the NuoL (ND5) subunit of the H+-translocating complex I, suggests the involvement of ShaA in cation transport. Bacillus subtilis ShaA contains six acidic residues that are conserved in ShaA homologues and are located in putative transmembrane helices. We examined the functional involvement of the six transmembrane acidic residues of ShaA by site-directed mutagenesis. Mutation in glutamate (Glu)-113 in TM-4, Glu-657 in TM-18, aspartate (Asp)-734 and Glu-747 in TM-20 abolished the antiport activity, suggesting that these residues play important roles in the ion transport of Sha. The acidic group was necessary and sufficient in Glu-657 and Asp-743, while it was not true of Glu-113 and Glu-747. Mutation in Asp-103 in TM-3, which is conserved in ShaA-types but not in ShaAB-types, partially affected on the antiport activity. Mutation in Asp-50 in TM-2 resulted in a unexpected phenotype: mutants retained the wild type level of ability to confer NaCl resistance to the Na+/H+ antiporter-deficient E. coli KNabc, but showed a very low antiport activity. The acidic group of Asp-50 and Asp-103 was not essential for the function. Our results suggested that these acidic residues are functionally involved in the ion transport of Sha, and some of them probably in cation binding and/or translocation.     

Comprehensive examination of charged intramembrane residues in a nucleoside transporter

Permeases of the equilibrative nucleoside transporter family mediate the uptake of nucleosides and/or nucleobases in a diverse array of eukaryotes and transport a host of drugs used for treatment of cancer, heart disease, AIDS, and parasitic infections. To identify residues that play central roles in transport function, we have systematically substituted by site-directed mutagenesis all the charged residues located within predicted transmembrane domains of the Leishmania donovani nucleoside transporter 1.1, LdNT1.1, which transports adenosine and the pyrimidine nucleosides. Substitution of three of these ten residues by uncharged amino acids resulted in loss of >95% transport activity, and we hence designated them "key" residues. These amino acids were Glu94, Lys153, and Arg404 located in transmembrane domains 2, 4, and 9, respectively. In addition, previous studies on the related LdNT2 inosine/guanosine transporter identified the highly conserved Asp389 and Arg393 (equivalent to Asp374 and Arg378 in LdNT1.1) in transmembrane domain 8 as key residues. Among these residues, the mutants in Arg393 (LdNT2) and Arg404 were strongly impaired in trafficking to the plasma membrane, but the other mutants were expressed with high to moderate efficiency at the cell surface, indicating that their mutation impaired transport activity per se. A conservative K153R substitution exhibited a change in substrate specificity, acquiring the ability to transport inosine, a nucleoside that is not a substrate for the wild-type LdNT1.1 permease. These results imply that the Glu94, Lys153, and Asp374 residues may play central roles in the mechanism of substrate translocation in LdNT1.1.     

Identification of structural and catalytic classes of highly conserved amino acid residues in lysine 2,3-aminomutase

Lysine 2,3-aminomutase (LAM) from Clostridium subterminale SB4 catalyzes the interconversion of (S)-lysine and (S)-beta-lysine by a radical mechanism involving coenzymatic actions of S-adenosylmethionine (SAM), a [4Fe-4S] cluster, and pyridoxal 5'-phosphate (PLP). The enzyme contains a number of conserved acidic residues and a cysteine- and arginine-rich motif, which binds iron and sulfide in the [4Fe-4S] cluster. The results of activity and iron, sulfide, and PLP analysis of variants resulting from site-specific mutations of the conserved acidic residues and the arginine residues in the iron-sulfide binding motif indicate two classes of conserved residues of each type. Mutation of the conserved residues Arg134, Asp293, and Asp330 abolishes all enzymatic activity. On the basis of the X-ray crystal structure, these residues bind the epsilon-aminium and alpha-carboxylate groups of (S)-lysine. However, among these residues, only Asp293 appears to be important for stabilizing the [4Fe-4S] cluster. Members of a second group of conserved residues appear to stabilize the structure of LAM. Mutations of arginine 130, 135, and 136 and acidic residues Glu86, Asp165, Glu236, and Asp172 dramatically decrease iron and sulfide contents in the purified variants. Mutation of Asp96 significantly decreases iron and sulfide content. Arg130 or Asp172 variants display no detectable activity, whereas variants mutated at the other positions display low to very low activities. Structural roles are assigned to this latter class of conserved amino acids. In particular, a network of hydrogen bonded interactions of Arg130, Glu86, Arg135, and the main chain carbonyl groups of Cys132 and Leu55 appears to stabilize the [4Fe-4S] cluster.     

Identification of conserved polar residues important for salt tolerance by the Na+/H+ exchanger of Schizosaccharomyces pombe

The Na+/H+ exchanger is a ubiquitous protein that transports Na+ and H+ in opposite directions across cell membranes. In fission yeast, the Na+/H+ exchanger sod2 plays a major role in the removal of excess detrimental intracellular sodium. The effect of mutagenesis of conserved polar amino acids of sod2 was examined by expressing 10 different mutant forms of sod2 in sod2 deficient S. pombe and characterizing salt tolerance. Asp145, 266, 267, and Glu173 were critical for proper function of sod2. Asp241 had an intermediate effect on sod2 function while mutation of Asp178 did not impair sod2 function. Simultaneous mutation of the Asp266, 267 pair impaired sod2 function. Mutation of each individual residue demonstrated that both were critical for sod2 function. Conservative mutations (Asp to Glu) of Asp266 and 267 failed to restore sod2 function. The results suggest that acidic residues associated with transmembrane segments are important in function, possibly being important in binding and coordinating cations.     

Identification of functionally important amino acid residues in Zymomonas mobilis levansucrase

The catalytic residues of levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) from Zymomonas mobilis were analyzed by random mutation and site-directed mutagenesis. We found that substitution of Glu278 with Asp and His reduced the k(cat) for sucrose hydrolysis 30- and 210-fold, respectively, strongly suggesting Glu278 plays a key role in catalyzing this reaction. Given the likelihood that another acidic amino residue was also involved, we constructed variants in which acidic amino acids located within homologous regions among bacterial levansucrases and fructosyltransferases were substituted, and found that substitution of Asp194, located in homologous region III, abolished sucrose hydrolysis. In addition, Glu278 was determined to be situated within the DXXER motif in homologous region IV conserved among bacterial levansucrases and fructosyltransferases, while Asp194 was within the triplet RDP motif conserved among bacterial levansucrases, fructosyltransferases and fructofuranosidases. Finally, comparison of our findings with published data on other site-directed mutated enzymes indicated His296, also located in homologous region IV, is crucial for catalysis of the transfructosylation reaction.     

A mechanism for the evolution of phosphorylation sites

Protein phosphorylation provides a mechanism for the rapid, reversible control of protein function. Phosphorylation adds negative charge to amino acid side chains, and negatively charged amino acids (Asp/Glu) can sometimes mimic the phosphorylated state of a protein. Using a comparative genomics approach, we show that nature also employs this trick in reverse by evolving serine, threonine, and tyrosine phosphorylation sites from Asp/Glu residues. Structures of three proteins where phosphosites evolved from acidic residues (DNA topoisomerase II, enolase, and C-Raf) show that the relevant acidic residues are present in salt bridges with conserved basic residues, and that phosphorylation has the potential to conditionally restore the salt bridges. The evolution of phosphorylation sites from glutamate and aspartate provides a rationale for why phosphorylation sometimes activates proteins, and helps explain the origins of this important and complex process.     

Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa

The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on Pseudomonas aeruginosa. The MexB subunit traverses the inner membrane 12 times and has two, two, and one charged residues in putative transmembrane segments 2 (TMS-2), TMS-4, and TMS-10, respectively. All five residues were mutated, and MexB function was evaluated by determining the MICs of antibiotics and fluorescent dye efflux. Replacement of Lys342 with Ala, Arg, or Glu and Glu346 with Ala, Gln, or Asp in TMS-2 did not have a discernible effect. Ala, Asn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led to loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibited only marginal function, suggesting that the length of the side chain at this position is important. The only replacements for Asp408 in TMS-4 or Lys939 in TMS-10 that exhibited significant function were Glu and Arg, respectively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost function. An Asp408-->Glu/Lys939-->Arg mutant retained significant activity, while an Asp407-->Glu/Lys939-->Arg mutant exhibited only marginal function. An Asp407-->Glu/Asp408-->Glu double mutant also lost activity, but significant function was restored by replacing Lys939 with Arg (Asp407-->Glu/Asp408-->Glu/Lys939-->Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp407, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 that is important for proton translocation and/or energy coupling.     

Alanine scanning mutagenesis of oxygen-containing amino acids in the transmembrane region of the Na,K-ATPase.

Oxygen-containing amino acids in the transmembrane region of the Na, K-ATPase alpha subunit were studied to identify residues involved in Na+ and/or K+ coordination by the enzyme. Conserved residues located in the polar face of transmembrane helices were selected using helical wheel and topological models of the enzyme. Alanine substitution of these residues were introduced into an ouabain-resistant sheep alpha1 isoform and expressed in HeLa cells. The capacity to generate essential Na+ and K+ gradients and thus support cell growth was used as an initial indication of the functionality of heterologous enzymes. Enzymes carrying alanine substitution of Ser94, Thr136, Ser140, Gln143, Glu144, Glu282, Thr334, Thr338, Thr340, Ser814, Tyr817, Glu818, Glu821, Ser822, Gln854, and Tyr994 supported cell growth, while those carrying substitutions Gln923Ala, Thr955Ala, and Asp995Ala did not. To study the effects of these latter replacements on cation binding, they were introduced into the wild-type alpha1 sheep isoform and expressed in mouse NIH3T3 cells where [3H]ouabain binding was utilized to probe the heterologous proteins. These substitutions did not affect ouabain, K+, or Na+ binding. Expression levels of these enzymes were similar to that of control. However, the level of Gln923Ala-, Thr955Ala-, or Asp995Ala-substituted enzyme at the plasma membrane was significantly lower than that of the wild-type isoform. Thus, these substitutions appear to impair the maturation process or targeting of the enzyme to the plasma membrane, but not cation-enzyme interactions. These results complete previous studies which have identified Ser755, Asp804, and Asp808 as absolutely essential for Na+ and K+ transport by the enzyme. Thus, it is significant that most transmembrane conserved-oxygen-containing residues in the Na,K-ATPase can be replaced without substantially affecting cation-enzyme interactions to the extent of preventing enzyme function. Consequently, other chemical groups, aromatic rings or backbone carbonyls, should be considered in models of cation-binding sites.     

A single amino acid in the hypervariable stem domain of vertebrate alpha1,3/1,4-fucosyltransferases determines the type 1/type 2 transfer. Characterization of acceptor substrate specificity of the lewis enzyme by site-directed mutagenesis

Alignment of 15 vertebrate alpha1,3-fucosyltransferases revealed one arginine conserved in all the enzymes employing exclusively type 2 acceptor substrates. At the equivalent position, a tryptophan was found in FUT3-encoded Lewis alpha1,3/1,4-fucosyltransferase (Fuc-TIII) and FUT5-encoded alpha1,3/1,4-fucosyltransferase, the only fucosyltransferases that can also transfer fucose in alpha1, 4-linkage. The single amino acid substitution Trp111 --> Arg in Fuc-TIII was sufficient to change the specificity of fucose transfer from H-type 1 to H-type 2 acceptors. The additional mutation of Asp112 --> Glu increased the type 2 activity of the double mutant Fuc-TIII enzyme, but the single substitution of the acidic residue Asp112 in Fuc-TIII by Glu decreased the activity of the enzyme and did not interfere with H-type 1/H-type 2 specificity. In contrast, substitution of Arg115 in bovine futb-encoded alpha1, 3-fucosyltransferase (Fuc-Tb) by Trp generated a protein unable to transfer fucose either on H-type 1 or H-type 2 acceptors. However, the double mutation Arg115 --> Trp/Glu116 --> Asp of Fuc-Tb slightly increased H-type 1 activity. The acidic residue adjacent to the candidate amino acid Trp/Arg seems to modulate the relative type 1/type 2 acceptor specificity, and its presence is necessary for enzyme activity since its substitution by the corresponding amide inactivated both Fuc-TIII and Fuc-Tb enzymes.     

Sterol methyltransferase: functional analysis of highly conserved residues by site-directed mutagenesis

Sterol methyltransferase (SMT), the enzyme from Saccharomyces cerevisiae that catalyzes the conversion of sterol acceptor in the presence of AdoMet to C-24 methylated sterol and AdoHcy, was analyzed for amino acid residues that contribute to C-methylation activity. Site-directed mutagenesis of nine aspartate or glutamate residues and four histidine residues to leucine (amino acids highly conserved in 16 different species) and expression of the resulting mutant proteins in Escherichia coli revealed that residues at H90, Asp125, Asp152, Glu195, and Asp276 are essential for catalytic activity. Each of the catalytically impaired mutants bound sterol, AdoMet, and 25-azalanosterol, a high energy intermediate analogue inhibitor of C-methylation activity. Changes in equilibrium binding and kinetic properties of the mutant enzymes indicated that residues required for catalytic activity are also involved in inhibitor binding. Analysis of the pH dependence of log kcat/Km for the wild-type SMT indicated a pH optimum for activity between 6 and 9. These results and data showing that only the mutant H90L binds sterol, AdoMet, and inhibitor to similar levels as the wild-type enzyme suggest that H90 may act as an acceptor in the coupled methylation-deprotonation reaction. Circular dichroism spectra and chromatographic information of the wild-type and mutant enzymes confirmed retention of the overall conformation of the enzyme during the various experiments. Taken together, our studies suggest that the SMT active center is composed of a set of acidic amino acids at positions 125, 152, 195, and 276, which contribute to initial binding of sterol and AdoMet and that the H90 residue functions subsequently in the reaction progress to promote product formation.     

Role of glutamate residues in substrate recognition by human MATE1 polyspecific H+/organic cation exporter

Human multidrug and toxic compound extrusion 1 (hMATE1) is an electroneutral H(+)/organic cation exchanger responsible for the final excretion step of structurally unrelated toxic organic cations in kidney and liver. To elucidate the molecular basis of the substrate recognition by hMATE1, we substituted the glutamate residues Glu273, Glu278, Glu300, and Glu389, which are conserved in the transmembrane regions, for alanine or aspartate and examined the transport activities of the resulting mutant proteins using tetraethylammonium (TEA) and cimetidine as substrates after expression in human embryonic kidney 293 (HEK-293) cells. All of these mutants except Glu273Ala were fully expressed and present in the plasma membrane of the HEK-293 cells. TEA transport activity in the mutant Glu278Ala was completely absent. Both Glu300Ala and Glu389Ala and all aspartate mutants exhibited significantly decreased activity. Glu273Asp showed higher affinity for cimetidine, whereas it has reduced affinity to TEA. Glu278Asp showed decreased affinity to cimetidine. Both Glu300Asp and Glu389Asp had lowered affinity to TEA, whereas the affinity of Glu389Asp to cimetidine was fourfold higher than that of the wild-type transporter with about a fourfold decrease in V(max) value. Both Glu273Asp and Glu300Asp had altered pH dependence for TEA uptake. These results suggest that all of these glutamate residues are involved in binding and/or transport of TEA and cimetidine but that their individual roles are different.     

Chicken mitochondrial cytochrome C oxidase subunit II: comparative analysis among the vertebrates

The chicken cytochrome c oxidase subunit II (COII) was cloned and sequenced. A comparison of the deduced chicken COII sequence with 4 other vertebrate counterparts revealed 64-66% amino acid sequence homology and 68-70% nucleotide sequence homology. Four peptide segments each of nine amino acids long are highly conserved across the 5 species. A redox-center was formed by three of these highly conserved domains, which include two invariant Cys and two invariant His residues for copper ion coordination, three strictly conserved Glu or Asp residues for cytochrome c binding, and highly conserved aromatic acid residues for electron transfer.     

Functional consequences of glutamate, aspartate, glutamine, and asparagine mutations in the stalk sector of the Ca2+-ATPase of sarcoplasmic reticulum

Nucleotides encoding glutamate, glutamine, aspartate, or asparagine residues within the stalk sector of the sarcoplasmic reticulum Ca2+-ATPase were altered by oligonucleotide-directed site-specific mutagenesis. The mutant cDNAs were expressed in COS-1 cells, and mutant Ca2+-ATPases were assayed for Ca2+ transport function and phosphoenzyme formation. Multiple mutations introduced into stalks, 1, 2, and 3 resulted in partial loss of Ca2+ transport function. In most cases, subsequent mutation of individual amino acids in the cluster had no effect on Ca2+ transport activity. In one cluster, however, it was possible to assign the reduction in Ca2+ transport activity to alterations of Asn111 and Asn114. The mutant Asn114 to alanine retained about 50% activity, whereas the change Asn111 to alanine retained only 10% activity. None of the mutations affected phosphorylation of the enzyme by ATP in the presence of Ca2+ or by inorganic phosphate in the absence of Ca2+. The combined experiments suggest that the reduced Ca2+ uptake observed in the Asn111 and Asn114 mutants was not due to a defect in enzyme activation by Ca2+ or in formation of the phosphorylated enzyme intermediate but rather to incompetent handling of the bound Ca2+ following ATP utilization. These results demonstrate that the acidic and amidated residues within the stalk region do not constitute the high affinity Ca2+-binding sites whose occupancy is required for enzyme activation. They may, however, act to sequester cytoplasmic Ca2+ and to channel it to domains that are involved in enzyme activation and cation translocation. Simultaneous mutation of 4 glutamate residues to alanine in the lumenal loop between transmembrane sequences M1 and M2 did not affect Ca2+ transport activity, indicating that acidic residues in this lumenal loop do not play an essential role in Ca2+ transport. Similarly, mutation of Glu192 and Asp196 in the beta-strand domain between stalk helices 2 and 3 did not affect Ca2+ transport activity, although mutation of Asp196 did diminish expression of the protein.     

Metal-tetracycline/H+ antiporter of Escherichia coli encoded by a transposon, Tn10. The role of the conserved dipeptide, Ser65-Asp66, in tetracycline transport

The transposon Tn10-encoded tetracycline resistance protein functions as a metal-tetracycline/H+ antiporter (Yamaguchi, A., Udagawa, T., and Sawai, T. (1990) J. Biol. Chem. 265, 4809-4813). The Ser65-Asp66 dipeptide is conserved in all known tetracycline antiporter proteins and is an important target for site-directed mutagenesis. When Asp66 was replaced by Asn, the transport activity was completely lost, whereas when it was replaced by Glu, the activity was reduced to 10% of the wild-type level, indicating that a negative charge at position 66 is essential for tetracycline transport. Replacement of Ser65 by Cys or Ala, in contrast, caused only a minor change in tetracycline transport activity. However, the Cys65 mutant antiporter was sensitive to sulfhydryl reagents. Complete inactivation of the Cys65 antiporter by N-ethylmaleimide was not prevented by the substrate. A less bulky reagent, methyl methanethiosulfonate, caused partial inactivation of the Cys65 antiporter without changing its affinity to the substrate. These results indicate that a region including the dipeptide plays an important role in metal-tetracycline transport except for substrate binding. It may act as a gate which opens on the charge-charge interaction between Asp66 and the metal-tetracycline.     

Functional consequences of mutations of conserved amino acids in the beta-strand domain of the Ca2(+)-ATPase of sarcoplasmic reticulum

The sequences Thr-Gly-Glu-Ser184 and Asp-Gln-Ser178 and individual residues Asp149, Asp157, and Asp162 in the sarcoplasmic reticulum Ca2(+)-ATPase are highly conserved throughout the family of cation-transporting ATPases. Mutant Thr181----Ala, Gly182----Ala, Glu183----Ala, and Glu183----Gln, created by in vitro mutagenesis, were devoid of Ca2+ transport activity. None of these mutations, however, affected phosphorylation of the enzyme by ATP in the presence of Ca2+ or by inorganic phosphate in the absence of Ca2+, indicating that the high affinity Ca2(+)-binding sites and the nucleotide-binding sites were intact. In each of these mutants, the ADP-sensitive phosphoenzyme intermediate (E1P) decayed to the ADP-insensitive form (E2P) very slowly relative to the wild-type enzyme, whereas E2P decayed at a rate similar to that of the wild-type enzyme. Thus, the inability of the mutants to transport Ca2+ was accounted for by an apparent block of the transport reaction at the E1P to E2P conformational transition. These results suggest that Thr181, Gly182, and Glu183 play essential roles in the conformational change between E1P and E2P. Mutation of Ser184, Asp157, or Ser178 had little or no effect on either Ca2+ transport activity or expression. Mutations of Asp149, Asp162, and Gln177, however, were poorly expressed. Where expression could be measured, in mutations to Asp162 and Gln177, Ca2+ transport activity was essentially equivalent to that of the wild-type enzyme.     

Identification of essential amino acid residues for catalytic activity and thermostability of novel chitosanase by site-directed mutagenesis

The functional importance of a conserved region in a novel chitosanase from Bacillus sp. CK4 was investigated. Each of the three carboxylic amino acid residues (Glu-50, Glu-62, and Asp-66) was changed to Asp and Gln or Asn and Glu by site-directed mutagenesis, respectively. The Asp-66-->Asn and Asp-66-->Glu mutation remarkably decreased kinetic parameters such as Vmax and kcat to approximately 1/1,000 those of the wild-type enzyme, indicating that the Asp-66 residue was essential for catalysis. The thermostable chitosanase contains three Cys residues at positions 49, 72, and 211. The Cys-49-->Ser/Tyr and Cys-72-->Ser/Tyr mutant enzymes were as stable to thermal inactivation and denaturating agents as the wild-type enzyme. However, the half-life of the Cys-211-->Ser/Tyr mutant enzyme was less than 10 min at 80 degrees C, while that of the wild-type enzyme was about 90 min. Moreover, the residual activity of Cys-211-->Ser/Tyr enzyme was substantially decreased by 8 M urea; and it lost all catalytic activity in 40% ethanol. These results show that the substitution of Cys with any amino acid residues at position 211 seems to affect the conformational stability of the chitosanase.