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Other Residues (other + residue)
Selected AbstractsRoles of adenine anchoring and ion pairing at the coenzyme B12 -binding site in diol dehydratase catalysisFEBS JOURNAL, Issue 24 2008Ken-ichi Ogura The X-ray structure of the diol dehydratase,adeninylpentylcobalamin complex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Ser,224, and of 6-NH2, N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the ,-NH2 group of Lys,135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing, Ser,224 and Lys,135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The S,224A, S,224N and K,135E mutants were 19,2% as active as the wild-type enzyme, whereas the K,135A, K,135Q and K,135R mutants retained 58,76% of the wild-type activity. The presence of a positive charge at the ,135 residue increased the affinity for cobalamins but was not essential for catalysis, and the introduction of a negative charge there prevented the enzyme,cobalamin interaction. The S,224A and S,224N mutants showed a kcat/kinact value that was less than 2% that of the wild-type, whereas for Lys,135 mutants this value was in the range 25,75%, except for the K,135E mutant (7%). Unlike the wild-type holoenzyme, the S,224N and S,224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Ser,224 is important for cobalt,carbon bond activation and for preventing the enzyme from being inactivated. Upon inactivation of the S,224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disappeared in EPR. 5,-Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism-based one. [source] Redox-regulated affinity of the third PDZ domain in the phosphotyrosine phosphatase PTP-BL for cysteine-containing target peptidesFEBS JOURNAL, Issue 13 2005Lieke C. J. Van Den Berk PDZ domains are protein,protein interaction modules that are crucial for the assembly of structural and signalling complexes. They specifically bind to short C-terminal peptides and occasionally to internal sequences that structurally resemble such peptide termini. The binding of PDZ domains is dominated by the residues at the P0 and P,2 position within these C-terminal targets, but other residues are also important in determining specificity. In this study, we analysed the binding specificity of the third PDZ domain of protein tyrosine phosphatase BAS-like (PTP-BL) using a C-terminal combinatorial peptide phage library. Binding of PDZ3 to C-termini is preferentially governed by two cysteine residues at the P,1 and P,4 position and a valine residue at the P0 position. Interestingly, we found that this binding is lost upon addition of the reducing agent dithiothrietol, indicating that the interaction is disulfide-bridge-dependent. Site-directed mutagenesis of the single cysteine residue in PDZ3 revealed that this bridge formation does not occur intermolecularly, between peptide and PDZ3 domain, but rather is intramolecular. These data point to a preference of PTP-BL PDZ3 for cyclic C-terminal targets, which may suggest a redox state-sensing role at the cell cortex. [source] Assessment of amino-acid substitutions at tryptophan 16 in ,-galactosidaseFEBS JOURNAL, Issue 5 2000Elizabeth Maranville The tryptophan residue at position 16 of coffee bean ,-galactosidase has previously been shown to be essential for enzyme activity. The potential role of this residue in the catalytic mechanism has been further studied by using site-directed mutagenesis to substitute every other amino acid for tryptophan at that site. Mutant enzymes were expressed in Pichia pastoris, a methylotrophic yeast strain, and their kinetic parameters were calculated. Only amino acids containing aromatic rings (phenylalanine and tyrosine) were able to support a significant amount of enzyme activity, but the kinetics and pH profiles of these mutants differed from wild-type. Substitution of arginine, lysine, methionine, or cysteine at position 16 allowed a small amount of enzyme activity with the optimal pH shifted towards more acidic. All other residues abolished enzyme activity. Our data support the hypothesis that tryptophan 16 is affecting the pKa of a carboxyl group at the active site that participates in catalysis. We also describe an assay for continuously measuring enzyme kinetics using fluorogenic 4-methylumbelliferyl substrates. This is useful in screening enzymes from colonies and determining the enzyme kinetics when the enzyme concentration is not known. [source] Identification of a triad of arginine residues in the active site of the ArsC arsenate reductase of plasmid R773FEMS MICROBIOLOGY LETTERS, Issue 2 2003Jin Shi Abstract ArsC from plasmid R773 catalyzes reduction of arsenate in Escherichia coli. Arg-60, Arg-94 and Arg-107 are near the active site residue Cys-12, suggesting that they form an anion binding pocket in the active site and/or participate in catalysis. These three arginine residues were altered to a variety of other residues by site-directed mutagenesis. Only mutants with arginine-to-lysine substitutions conferred arsenate resistance in vivo, although purified R60A, R60E, R60K exhibited varying levels of enzymatic activity. The data support the hypothesis that this triad of arginine residues is involved in arsenate binding and transition-state stabilization. [source] CxxS: Fold-independent redox motif revealed by genome-wide searches for thiol/disulfide oxidoreductase functionPROTEIN SCIENCE, Issue 10 2002Dmitri E. Fomenko Abstract Redox reactions involving thiol groups in proteins are major participants in cellular redox regulation and antioxidant defense. Although mechanistically similar, thiol-dependent redox processes are catalyzed by structurally distinct families of enzymes, which are difficult to identify by available protein function prediction programs. Herein, we identified a functional motif, CxxS (cysteine separated from serine by two other residues), that was often conserved in redox enzymes, but rarely in other proteins. Analyses of complete Escherichia coli, Campylobacter jejuni, Methanococcus jannaschii, and Saccharomyces cerevisiae genomes revealed a high proportion of proteins known to use the CxxS motif for redox function. This allowed us to make predictions in regard to redox function and identity of redox groups for several proteins whose function previously was not known. Many proteins containing the CxxS motif had a thioredoxin fold, but other structural folds were also present, and CxxS was often located in these proteins upstream of an ,-helix. Thus, a conserved CxxS sequence followed by an ,-helix is typically indicative of a redox function and corresponds to thiol-dependent redox sites in proteins. The data also indicate a general approach of genome-wide identification of redox proteins by searching for simple conserved motifs within secondary structure patterns. [source] Differential tetraethylammonium sensitivity of KCNQ1,4 potassium channelsBRITISH JOURNAL OF PHARMACOLOGY, Issue 3 2000J K Hadley In Shaker -group potassium channels the presence of a tyrosine residue, just downstream of the pore signature sequence GYG, determines sensitivity to tetraethylammonium (TEA). The KCNQ family of channels has a variety of amino acid residues in the equivalent position. We studied the effect of TEA on currents generated by KCNQ homomers and heteromers expressed in CHO cells. We used wild-type KCNQ1,4 channels and heteromeric KCNQ2/3 channels incorporating either wild-type KCNQ3 subunits or a mutated KCNQ3 in which tyrosine replaced threonine at position 323 (mutant T323Y). IC50 values were (mM): KCNQ1, 5.0; KCNQ2, 0.3; KCNQ3, >30; KCNQ4, 3.0; KCNQ2+KCNQ3, 3.8; and KCNQ2+KCNQ3(T323Y), 0.5. While the high TEA sensitivity of KCNQ2 may be conferred by a tyrosine residue lacking in the other channels, the intermediate TEA sensitivity of KCNQ1 and KCNQ4 implies that other residues are also important in determining TEA block of the KCNQ channels. British Journal of Pharmacology (2000) 129, 413,415; doi:10.1038/sj.bjp.0703086 [source] Alteration of the Diastereoselectivity of 3-Methylaspartate Ammonia Lyase by Using Structure-Based MutagenesisCHEMBIOCHEM, Issue 13 2009Hans Raj Abstract 3-Methylaspartate ammonia-lyase (MAL) catalyzes the reversible amination of mesaconate to give both (2S,3S)-3-methylaspartic acid and (2S,3R)-3-methylaspartic acid as products. The deamination mechanism of MAL is likely to involve general base catalysis, in which a catalytic base abstracts the C3 proton of the respective stereoisomer to generate an enolate anion intermediate that is stabilized by coordination to the essential active-site MgII ion. The crystal structure of MAL in complex with (2S,3S)-3-methylaspartic acid suggests that Lys331 is the only candidate in the vicinity that can function as a general base catalyst. The structure of the complex further suggests that two other residues, His194 and Gln329, are responsible for binding the C4 carboxylate group of (2S,3S)-3-methylaspartic acid, and hence are likely candidates to assist the MgII ion in stabilizing the enolate anion intermediate. In this study, the importance of Lys331, His194, and Gln329 for the activity and stereoselectivity of MAL was investigated by site-directed mutagenesis. His194 and Gln329 were replaced with either an alanine or arginine, whereas Lys331 was mutated to a glycine, alanine, glutamine, arginine, or histidine. The properties of the mutant proteins were investigated by circular dichroism (CD) spectroscopy, kinetic analysis, and 1H NMR spectroscopy. The CD spectra of all mutants were comparable to that of wild-type MAL, and this indicates that these mutations did not result in any major conformational changes. Kinetic studies demonstrated that the mutations have a profound effect on the values of kcat and kcat/KM; this implicates Lys331, His194 and Gln329 as mechanistically important. The 1H NMR spectra of the amination and deamination reactions catalyzed by the mutant enzymes K331A, H194A, and Q329A showed that these mutants have strongly enhanced diastereoselectivities. In the amination direction, they catalyze the conversion of mesaconate to yield only (2S,3S)-3-methylaspartic acid, with no detectable formation of (2S,3R)-3-methylaspartic acid. The results are discussed in terms of a mechanism in which Lys331, His194, and Gln329 are involved in positioning the substrate and in formation and stabilization of the enolate anion intermediate. [source] Design of peptides with branched ,-carbon dehydro-residues: syntheses, crystal structures and molecular conformations of two peptides, (I) N -Carbobenzoxy-,Val-Ala-Leu-OCH3 and (II) N -Carbobenzoxy-,Ile-Ala-Leu-OCH3CHEMICAL BIOLOGY & DRUG DESIGN, Issue 2 2003R. Vijayaraghavan Abstract: Highly specific structures can be designed by inserting dehydro-residues into peptide sequences. The conformational preferences of branched , -carbon residues are known to be different from other residues. As an implication it was expected that the branched , -carbon dehydro-residues would also induce different conformations when substituted in peptides. So far, the design of peptides with branched , -carbon dehydro-residues at (i + 1) position has not been reported. It may be recalled that the nonbranched , -carbon residues induced , -turn II conformation when placed at (i + 2) position while branched , -carbon residues induced , -turn III conformation. However, the conformation of a peptide with a nonbranched , -carbon residue when placed at (i + 1) position was not found to be unique as it depended on the stereochemical nature of its neighbouring residues. Therefore, in order to induce predictably unique structures with dehydro-residues at (i + 1) position, we have introduced branched , -carbon dehydro-residues instead of nonbranched , -carbon residues and synthesized two peptides: (I) N -Carbobenzoxy-,Val-Ala-Leu-OCH3 and (II) N -Carbobenzoxy-,Ile-Ala-Leu-OCH3 with ,Val and ,Ile, respectively. The crystal structures of peptides (I) and (II) have been determined and refined to R-factors of 0.065 and 0.063, respectively. The structures of both peptides were essentially similar. Both peptides adopted type II , -turn conformations with torsion angles; (I): ,1 = ,38.7 (4)°, ,1 = 126.0 (3)°; ,2 = 91.6 (3)°, ,2 = ,9.5 (4)° and (II): ,1 = ,37.0 (6)°, ,1 = 123.6 (4)°, ,2 = 93.4 (4), ,2 = ,11.0(4)° respectively. Both peptide structures were stabilized by intramolecular 4,1 hydrogen bonds. The molecular packing in both crystal structures were stabilized in each by two identical hydrogen bonds N1,O1, (,x, y + 1/2, ,z) and N2,O2, (,x + 1, y + 1/2, ,z) and van der Waals interactions. [source] | |||||||||||||