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.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis

Computer analysis of the crystallographic structure of the A subunit of Escherichia coil heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53 → Glu or Asp, Ser-63 → Lys, Val-97 → Lys, Tyr-104 → Lys or Asp, and Ser-14 → Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41 → Phe, Ala-45 → Tyr or Glu, Val-53 → Tyr, Val-60 → Gly, Ser-68 → Pro, His-70 → Pro, Val-97 → Tyr and Ser-114 → Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54 → Lys or Ala, Tyr-59 → Met, Ser-68 → Lys, Ala-72 → Arg, His or Asp and Arg-192 → Asn. The results provide a further understanding of the structure–function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Site-specific methionine sulfoxide formation is the structural basis of chromatographic heterogeneity of apolipoproteins A-I, C-II, and C-III.

ApoA-I and apoC-II are eluted in two isoforms and apoC-III2 is eluted in three isoforms by reversed phase high performance liquid chromatography (HPLC). The structural basis of these nongenetic heterogeneities was unravelled using HPLC of proteolytic peptides and time-of-flight secondary ion mass spectrometry (TOF-SIMS). In apoA-I, the chromatographic microheterogeneity was caused by the formation of methionine sulfoxides (MetSO). However, only residues Met112 and Met148 were found oxidized, whereas Met86 was unaffected and also resistant towards artificial oxidation. To assess whether and to what extent amino acid substitutions in apoA-I might affect methionine sulfoxidation, the tryptic peptides of 13 different mutant apoA-I proteins from 24 heterozygous apoA-I variant carriers were analyzed by HPLC. In normal apoA-I, the ratios MetSO112/Met112 and MetSO148/Met148 were highly variable. By contrast, the relative ratio of oxidation of methionine residues 112 and 148 was constant. The amino acid changes Lys107----Met, Lys107----O, Glu139----Gly, Glu147----Val, and Pro165----Arg resulted in the preferential oxidation of Met112, and Asp103----Asn resulted in a preferential oxidation of Met148; whereas Pro3----Arg, Pro3----His, Pro4----Arg, Asp89----Glu, Ala158----Asp, Glu198----Lys, and Asp213----Gly had no impact. ApoC-II and apoC-III isoforms differed by the oxidation of the two methionine residues in these proteins. Whereas in apoC-II both methionine residues were oxidized in parallel, in apoC-III the two methionine residues differed in their susceptibility towards oxidation. We conclude that the formation of MetSO depends on the molecular microenvironment within a protein.     

The function of Asp70, Glu77 and Lys79 in the Escherichia coli MutH protein

The MutH protein, which is part of the Dam-directed mismatch repair system of Escherichia coli, introduces nicks in the unmethylated strand of a hemi-methylated DNA duplex. The latent endonuclease activity of MutH is activated by interaction with MutL, another member of the repair system. The crystal structure of MutH suggested that the active site residues include Asp70, Glu77 and Lys79, which are located at the bottom of a cleft where DNA binding probably occurs. We mutated these residues to alanines and found that the mutant proteins were unable to complement a chromosomal mutH deletion. The purified mutant proteins were able to bind to DNA with a hemi-methylated GATC sequence but had no detectable endonuclease activity with or without MutL. Although the data are consistent with the prediction of a catalytic role for Asp70, Glu77 and Lys79, it cannot be excluded that they are also involved in binding to MutL.     

Three subunits contribute amino acids to the active site of tetrameric adenylosuccinate lyase: Lys268 and Glu275 are required

Tetrameric adenylosuccinate lyase (ASL) of Bacillus subtilis catalyzes the cleavage of adenylosuccinate to form AMP and fumarate. We previously reported that two distinct subunits contribute residues to each active site, including the His68 and His89 from one and His141 from a second subunit [Brosius, J. L., and Colman, R. F. (2000) Biochemistry 39, 13336-13343]. Glu(275) is 2.8 A from His141 in the ASL crystal structure, and Lys268 is also in the active site region; Glu275 and Lys268 come from a third, distinct subunit. Using site-directed mutagenesis, we have replaced Lys268 by Arg, Gln, Glu, and Ala, with specific activities of the purified mutant enzymes being 0.055, 0.00069, 0.00028, and 0.0, respectively, compared to 1.56 units/mg for wild-type (WT) enzyme. Glu275 was substituted by Gln, Asp, Ala, and Arg; none of these homogeneous mutant enzymes has detectable activity. Circular dichroism and light scattering reveal that neither the secondary structure nor the oligomeric state of the Lys268 mutant enzymes has been perturbed. Native gel electrophoresis and circular dichroism indicate that the Glu275 mutant enzymes are tetramers, but their conformation is altered slightly. For K268R, the K(m)s for all substrates are similar to WT enzyme. Binding studies using [2-3H]-adenylosuccinate reveal that none of the Glu275 mutant enzymes, nor inactive K268A, can bind substrate. We propose that Lys268 participates in binding substrate and that Glu275 is essential for catalysis because of its interaction with His141. Incubation of H89Q with K268Q or E275Q leads to restoration of up to 16% WT activity, while incubation of H141Q with K268Q or E275Q results in 6% WT activity. These complementation studies provide the first functional evidence that a third subunit contributes residues to each intersubunit active site of ASL. Thus, adenylosuccinate lyase has four active sites per enzyme tetramer, each of which is formed from regions of three subunits.     

Functional changes in a novel uracil-DNA glycosylase determined by mutational analyses

Uracil-DNA glycosylase (UDG) is a ubiquitous enzyme found in bacteria and eukaryotes, which removes uracil residues from DNA strands. Methanococcus jannaschii UDG (MjUDG), a novel monofunctional glycosylase, contains a helix-hairpin-helix (HhH) motif and a Gly/Pro rich loop (GPD region), which is important for catalytic activity; it shares these features with other glycosylases, such as endonuclease III. First, to examine the role of two conserved amino acid residues (Asp150 and Tyr152) in the HhH-GPD region of MjUDG, mutant MjUDG proteins were constructed, in which Asp150 was replaced with either Glu or Trp (D150E and D150W), and Tyr152 was replaced with either Glu or Asn (Y152E and Y152N). Mutant D150W completely lacked DNA glycosylase activity, whereas D150E displayed reduced activity of about 70% of the wild type value. However, the mutants Y152E and Y152N retained unchanged levels of UDG activity. We also replaced Glu132 in the HhH motif with a lysine residue equivalent to Lys120 in endonuclease III. This mutation converted the enzyme into a bifunctional glycosylase/AP lyase capable of both removing uracil at a glycosylic bond and cleaving the phosphodiester backbone at an AP site. Mutant E132K catalyzes a β-elimination reaction at the AP site via uracil excision and forms a Schiff base intermediate in the form of a protein-DNA complex. This text was submitted by the authors in English.     

Engineering of the pH-dependence of thermolysin activity as examined by site-directed mutagenesis of Asn112 located at the active site of thermolysin

Asn112 is located at the active site of thermolysin, 5-8 A from the catalytic Zn2+ and catalytic residues Glu143 and His231. When Asn112 was replaced with Ala, Asp, Glu, Lys, His, and Arg by site-directed mutagenesis, the mutant enzymes N112D and N112E, in which Asn112 is replaced with Asp and Glu, respectively, were secreted as an active form into Escherichia coli culture medium, while the other four were not. In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide, the kcat/Km values of N112D and N112E exhibited bell-shaped pH-dependence, as did the wild-type thermolysin (WT). The acidic pKa of N112D was 5.7 +/- 0.1, higher by 0.4 +/- 0.2 units than that of WT, suggesting that the introduced negative charge suppressed the protonation of Glu143 or Zn2+-OH. In the hydrolysis of a negatively charged substrate, N-carbobenzoxy-l-Asp-l-Phe methyl ester (ZDFM), the pH-dependence of kcat/Km of the mutants decreased with increase in pH from 5.5 to 8.5, while that of WT was bell-shaped. This difference might be explained by the electrostatic repulsion between the introduced Asp/Glu and ZDFM, suggesting that introducing ionizing residues into the active site of thermolysin might be an effective means of modifying its pH-activity profile.     

5'-methylthioadenosine nucleosidase from yellow lupine (Lupinus luteus): molecular characterization and mutational analysis

This is report of mutational analysis of higher plant 5'-methylthioadenosine nucleosidase (MTAN). We identified and characterized the gene encoding yellow lupine (Lupinus luteus) MTAN (LlMTAN). The role of active site amino acids residues Glu24, Phe134, Glu188 and Asp211 was analyzed by site-directed mutagenesis. The Glu24Gln and Asp211Asn substitutions completely abolished the enzyme activity. The Glu188Gln mutant showed only trace activity toward 5'-methylthioadenosine. These results indicate that these three amino acid residues are necessary for enzyme activity. Furthermore, as the result of replacement of Phe134 by less bulky leucine, LlMTAN acquired the ability to bind and hydrolyze S-adenosylhomocysteine. We also analyzed the sequence of the LlMTAN promoter region. It appeared that there may be a direct link between LlMTAN expression regulation and sulfate metabolism.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Engineering PQQ glucose dehydrogenase with improved substrate specificity. Site-directed mutagenesis studies on the active center of PQQ glucose dehydrogenase

Site-directed mutagenesis was carried out on the active site of water-soluble PQQ glucose dehydrogenase (PQQGDH-B) to improve its substrate specificity. Amino acid substitution of His168 resulted in a drastic decrease in the enzyme's catalytic activity, consistent with its putative catalytic role. Substitutions were also carried out in neighboring residues, Lys166, Asp167, and Gln169, in an attempt to alter the enzyme's substrate binding site. Lys166 and Gln169 mutants showed only minor changes in substrate specificity profiles. In sharp contrast, mutants of Asp167 showed considerably altered specificity profiles. Of the numerous Asp167 mutants characterized, Asp167Glu showed the best substrate specificity profile, while retaining most of its catalytic activity for glucose and stability. We also investigated the cumulative effect of combining the Asp167Glu substitution with the previously reported Asn452Thr mutation. Interpretation of the effect of the replacement of Asp167 to Glu on the alteration of substrate specificity in relation with the predicted 3D model of PQQGDH-B is also discussed.     

Modification of the substrate specificity of porcine pepsin for the enzymatic production of bovine hide gelatin

The substrate specificity of porcine pepsin has been altered by site-directed mutagenesis in an attempt to selectively cleave bovine hide collagen at only a few sites, similar to cathepsin D, for the production of high quality gelatin. Kinetic parameters were determined using chromogenic peptide substrates based on the sequence Lys-Pro-Xaa-Yaa-Phe*Nph-Arg-Leu (where Xaa is Ile or Pro, Yaa is Glu. Leu, Gln or Lys, Nph is p-nitrophenylalanine, and * is the site of cleavage). Substitution of Thr222 and Glu287 within the S2 subsite of pepsin by Val and Met, respectively, produced a double mutant with a two- to fourfold higher kcat/Km, compared with wild-type pepsin, for the chromogenic peptides with residues Leu, Gln, and Glu at position P2 (Yaa). The results suggest that the functional group of the P2 side chain may be exposed to solvent, while the aliphatic portion interacts with hydrophobic residues comprising S2. Wild-type pepsin cleaved a peptide corresponding to the carboxy-terminal telopeptide region of bovine type I collagen alpha1 chain, SGGYDLSFLPQPPQE, predominantly at three sites (Asp-Leu, Leu-Ser, and Phe-Leu) and at a significantly lower rate at Ser-Phe. However, Thr222Val/Glu287Met cleaved site Ser-Phe at a rate 20-fold higher than the wild-type. Significantly, enzymes containing the double substitution Phe111Thr/Leu112Phe cleaved this peptide predominantly at one site Leu-Ser (similar to cathepsin D) and at a rate 23-fold higher than the wild-type. These mutants can potentially enhance the rate of solubilization of bovine hide collagen under conditions mild enough to maintain the triple helix structure and hence minimize the rate of subsequent denaturation and proteolytic cleavage.     

Probing electrostatic interactions and ligand binding in aspartyl-tRNA synthetase through site-directed mutagenesis and computer simulations

Faithful genetic code translation requires that each aminoacyl-tRNA synthetase recognise its cognate amino acid ligand specifically. Aspartyl-tRNA synthetase (AspRS) distinguishes between its negatively-charged Asp substrate and two competitors, neutral Asn and di-negative succinate, using a complex network of electrostatic interactions. Here, we used molecular dynamics simulations and site-directed mutagenesis experiments to probe these interactions further. We attempt to decrease the Asp/Asn binding free energy difference via single, double and triple mutations that reduce the net positive charge in the active site of Escherichia coli AspRS. Earlier, Glutamine 199 was changed to a negatively-charged glutamate, giving a computed reduction in Asp affinity in good agreement with experiment. Here, Lysine 198 was changed to a neutral leucine; then, Lys198 and Gln199 were mutated simultaneously. Both mutants are predicted to have reduced Asp binding and improved Asn binding, but the changes are insufficient to overcome the initial, high specificity of the native enzyme, which retains a preference for Asp. Probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments, we found no detectable activity for the mutant enzymes, indicating weaker Asp binding and/or poorer transition state stabilization. The simulations show that the mutations' effect is partly offset by proton uptake by a nearby histidine. Therefore, we performed additional simulations where the nearby Histidines 448 and 449 were mutated to neutral or negative residues: (Lys198Leu, His448Gln, His449Gln), and (Lys198Leu, His448Glu, His449Gln). This led to unexpected conformational changes and loss of active site preorganization, suggesting that the AspRS active site has a limited structural tolerance for electrostatic modifications. The data give insights into the complex electrostatic network in the AspRS active site and illustrate the difficulty in engineering charged-to-neutral changes of the preferred ligand.     

Mutation of active site residues in synthetic T4-lysozyme gene and their effect on lytic activity

The active site amino acids (Glu11 and Asp20) in T4-lysozyme have been mutated to their isosteric residues Gln or Asn and/or acidic residues such as Glu----Asp or Asp----Glu by the oligonucleotide-replacement method. Out of eight mutants so generated the mutant T4-lysozyme obtained from pTLY.Asp11 retains maximum amount of activity (approximately 16%), pTLY.Asn20 the least (0.9%) whereas pTLY.Gln11 lost completely. A systematic study of the active and inactive mutants thus generated supports the important role of Glu11 and Asp20 in T4-lysozyme activity as predicted in earlier studies.     

Different sensitivities of mutants and chimeric forms of human muscle and liver fructose-1,6-bisphosphatases towards AMP

AMP is an allosteric inhibitor of human muscle and liver fructose-1,6-bisphosphatase (FBPase). Despite strong similarity of the nucleotide binding domains, the muscle enzyme is inhibited by AMP approximately 35 times stronger than liver FBPase: I0.5 for muscle and for liver FBPase are 0.14 microM and 4.8 microM, respectively. Chimeric human muscle (L50M288) and chimeric human liver enzymes (M50L288), in which the N-terminal residues (1-50) were derived from the human liver and human muscle FBPases, respectively, were inhibited by AMP 2-3 times stronger than the wild-type liver enzyme. An amino acid exchange within the N-terminal region of the muscle enzyme towards liver FBPase (Lys20-->Glu) resulted in 13-fold increased I0.5 values compared to the wild-type muscle enzyme. However, the opposite exchanges in the liver enzyme (Glu20-->Lys and double mutation Glu19-->Asp/Glu20-->Lys) did not change the sensitivity for AMP inhibition of the liver mutant (I0.5 value of 4.9 microM). The decrease of sensitivity for AMP of the muscle mutant Lys20-->Glu, as well as the lack of changes in the inhibition by AMP of liver mutants Glu20-->Lys and Glu19-->Asp/Glu20-->Lys, suggest a different mechanism of AMP binding to the muscle and liver enzyme.     

Mechanistic implications from crystalline complexes of wild-type and mutant adenylosuccinate synthetases from Escherichia coli.

Asp13 and His41 are essential residues of adenylosuccinate synthetase, putatively catalyzing the formation of adenylosuccinate from an intermediate of 6-phosphoryl-IMP. Wild-type adenylosuccinate synthetase and three mutant synthetases (Arg143 --> Leu, Lys16 --> Gln, and Arg303 --> Leu) from Eschericha coli have been crystallized in the presence of IMP, hadacidin (an analogue of L-aspartate), Mg2+, and GTP. The active site of each complex contains 6-phosphoryl-IMP, Mg2+, GDP, and hadacidin, except for the Arg303 --> Leu mutant, which does not bind hadacidin. In response to the formation of 6-phosphoryl-IMP, Asp13 enters the inner coordination sphere of the active site Mg2+. His41 hydrogen bonds with 6-phosphoryl-IMP, except in the Arg303 --> Leu complex, where it remains bound to the guanine nucleotide. Hence, recognition of the active site Mg2+ by Asp13 evidently occurs after the formation of 6-phosphoryl-IMP, but recognition of the intermediate by His41 may require the association of L-aspartate with the active site. Structures reported here support a mechanism in which Asp13 and His41 act as the catalytic base and acid, respectively, in the formation of 6-phosphoryl-IMP, and then act together as catalytic acids in the subsequent formation of adenylosuccinate.     

Functional consequences of alterations to amino acids located in the hinge domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis of the sarcoplasmic reticulum Ca(2+)-ATPase was used to investigate the functional roles of 18 amino acid residues located at or near the "hinge-domain," a highly conserved region of the cation-transporting ATPases. Mutation of Lys684 to arginine, alanine, histidine, and glutamine resulted in complete loss of calcium transport function and ATPase activity. For the Lys684----Ala, histidine, and glutamine mutants, this coincided with a loss of the ability to form a phosphorylated intermediate from ATP or Pi. The Lys684----Arg mutant retained the ability to phorphorylate from ATP with normal apparent affinity, demonstrating the importance of the positive charge. On the other hand, no phosphorylation was observed with Pi as substrate in this mutant. Examination of the partial reactions after phosphorylation from ATP in the Lys684----Arg mutant demonstrated a reduction of the rate of transformation of the ADP-sensitive phosphoenzyme intermediate (E1P) to the ADP-insensitive phosphoenzyme intermediate (E2P), which could account for the loss of transport function. Once accumulated, the E2P intermediate was able to decompose rapidly in the presence of K+ at neutral pH. These results may be interpreted in terms of a preferential destabilization of protein phosphate interactions in the E2P form of this mutant. The Asp703----Ala and Asn-Asp707----Ala-Ala mutants were completely inactive and unable to form phosphoenzyme intermediates from ATP or Pi. In these mutants as well as in the Lys684----Ala mutant, nucleotides were found to protect with normal affinity against intramolecular cross-linking induced with glutaraldehyde, indicating that the nucleotide binding site was intact. Mutation of Glu646, Glu647, Asp659, Asp660, Glu689, Asp695, Glu696, Glu715, and Glu732 to alanine did not affect the maximum rates of calcium transport and ATP hydrolysis or the apparent affinities for calcium and ATP. Mutation of the 2 highly conserved proline residues, Pro681 and Pro709, as well as Lys728, to alanine resulted in partially inhibited Ca(2+)-ATPase enzymes with retention of the ability to form a phosphoenzyme intermediate from ATP or Pi and with normal apparent affinities for ATP and calcium. The proline mutants retained the biphasic ATP concentration dependence of ATPase activity, characteristic of the wild-type, and therefore the partial inhibition of turnover could not be ascribed to a disruption of the low affinity modulatory ATP site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enzymatic properties of mutant Escherichia coli tryptophan synthase alpha-subunits

Thirty-nine mutant tryptophan synthase alpha subunits have been purified and analyzed (in the presence of the beta 2-subunit) for their enzymatic (kcat, Km) behavior in the reactions catalyzed by the alpha 2.beta 2 complex, the fully constituted form of this enzyme. The mutant alpha subunits, obtained by in vitro random, saturation mutagenesis of the encoding trpA gene, contain single amino acid substitutions at sites within the first 121 residues of the alpha polypeptide. Four categories of altered residues have been tentatively assigned roles in the catalytic functions of this enzyme: 1) catalytic residues (Glu49 and Asp60); 2) residues involved in substrate binding or orientation (Phe22, Thr63, Gln65, Tyr102, and Leu105); 3) residues involved in alpha.beta subunit interactions (Gly51, Pro53, Asp56, Asp60, Pro62, Ala67, Phe72, Thr77, Pro78, Tyr102, Asn104, Leu105, and Asn108); and 4) residues with no apparent catalytic roles. Catalytic residue alterations result in no detectable activity in the alpha-subunit specific reactions. Substrate binding/orientation roles are detected enzymatically primarily as rate defects; alterations only at Tyr102 result in apparent Km effects. alpha.beta interaction roles are detected as rate defects in all tryptophan synthase reactions plus Km increases for the alpha-subunit substrate, indole-3-glycerol phosphate, only when L-serine is present at the beta 2-subunit active site. A substitution at only one site, Asn104, appears to be unique in its potential effect on intersubunit channeling of indole, the product of the alpha-subunit specific reaction, to the beta 2-subunit active site.