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1.
Supangat S Park SO Seo KH Lee SY Park YS Lee KH 《Acta biochimica et biophysica Sinica》2008,40(6):513-518
Sepiapterin reductase from Chlorobium tepidum (cSR) catalyzes the synthesis of a distinct tetrahydrobiopterin (BH4), L -threo-BH4, different from the mammalian enzyme product. The 3-D crystal structure of cSR has revealed that the product configuration is determined solely by the substrate binding mode within the well-conserved catalytic triads. In cSR, the sepiapterin is stacked between two aromatic side chains of Phe-99 and Trp-196 and rotated approximately 180° around the active site from the position in mouse sepiapterin reductase. To confirm their roles in substrate binding, we mutated Phe-99 and/or Trp-196 to alanine (F99A, W196A) by site-directed mutagenesis and comparatively examined substrate binding of the purified proteins by kinetics analysis and differential scanning calorimetry. These mutants had higher K m values than the wild type. Remarkably, the W196A mutation resulted in a higher K m increase compared with the F99A mutation. Consistent with the results, the melting temperature ( T m ) in the presence of sepiapterin was lower in the mutant proteins and the worst was W196A. These findings indicate that the two residues are indispensable for substrate binding in cSR, and Trp-196 is more important than Phe-99 for different stereoisomer production. 相似文献
2.
Roles of aromatic residues in high interfacial activity of Naja naja atra phospholipase A2 总被引:2,自引:0,他引:2
Acidic phospholipase A2 (PLA2) from the venom of Chinese cobra (Naja naja atra) has high activity on zwitterionic membranes and contains six aromatic residues, including Tyr-3, Trp-18, Trp-19, Trp-61, Phe-64, and Tyr-110, on its putative interfacial binding surface. To assess the roles of these aromatic residues in the interfacial catalysis of N. n. atra PLA2, we mutated them to Ala and measured the effects on its interfacial catalysis. Enzymatic activities of the mutants toward various vesicle substrates and human neutrophils indicate that all but Trp-18 make significant contributions to interfacial catalysis. Among these aromatic residues, Trp-19, Trp-61, and Phe-64 play the most important roles. Binding affinities of the mutants for phospholipid-coated beads and their monolayer penetration indicate that Trp-19, Trp-61, and Phe-64 are critically involved in interfacial binding of N. n. atra PLA2 and penetrate into the membrane during the interfacial catalysis of N. n. atra PLA2. Further thermodynamic analysis suggests that the side chain of Phe-64 is fully inserted into the hydrophobic core of membrane whereas those of Trp-19 and Trp-61 are located in the membrane-water interface. Together, these results show that all three types of aromatic residues can play important roles in interfacial binding of PLA2 depending on their location and side-chain orientation. They also indicate that these aromatic side chains interact with membranes in distinct modes because of their different intrinsic preference for different parts of membranes. 相似文献
3.
S Ghosh D Wolan S Adak B R Crane N S Kwon J A Tainer E D Getzoff D J Stuehr 《The Journal of biological chemistry》1999,274(34):24100-24112
Inducible nitric-oxide synthase (iNOS) is a hemeprotein that requires tetrahydrobiopterin (H4B) for activity. The influence of H4B on iNOS structure-function is complex, and its exact role in nitric oxide (NO) synthesis is unknown. Crystal structures of the mouse iNOS oxygenase domain (iNOSox) revealed a unique H4B-binding site with a high degree of aromatic character located in the dimer interface and near the heme. Four conserved residues (Arg-375, Trp-455, Trp-457, and Phe-470) engage in hydrogen bonding or aromatic stacking interactions with the H4B ring. We utilized point mutagenesis to investigate how each residue modulates H4B function. All mutants contained heme ligated to Cys-194 indicating no deleterious effect on general protein structure. Ala mutants were monomers except for W457A and did not form a homodimer with excess H4B and Arg. However, they did form heterodimers when paired with a full-length iNOS subunit, and these were either fully or partially active regarding NO synthesis, indicating that preserving residue identities or aromatic character is not essential for H4B binding or activity. Aromatic substitution at Trp-455 or Trp-457 generated monomers that could dimerize with H4B and Arg. These mutants bound Arg and H4B with near normal affinity, but Arg could not displace heme-bound imidazole, and they had NO synthesis activities lower than wild-type in both homodimeric and heterodimeric settings. Aromatic substitution at Phe-470 had no significant effects. Together, our work shows how hydrogen bonding and aromatic stacking interactions of Arg-375, Trp-457, Trp-455, and Phe-470 influence iNOSox dimeric structure, heme environment, and NO synthesis and thus help modulate the multiple effects of H4B. 相似文献
4.
Numerous polypeptides and proteins form amyloid deposits in vivo or in vitro. The mechanism of amyloid formation is not well-understood particularly in the case where unstructured polypeptides assemble to form amyloid. Aromatic-aromatic interactions are known to be important in globular proteins, and the possibility that they might play a key role in amyloid formation has been raised. The results of Ala-scanning experiments on short polypeptides derived from Amylin have suggested that aromatic interactions could be particularly important for this system. Here, we examine a set of Amylin-derived polypeptides in which the single aromatic residue has been substituted with a Leu and Ala. A peptide corresponding to residues 21-29 with a Phe-23 to Leu substitution, a free N terminus, and amidated C terminus readily forms amyloid. Shorter peptides derived from the putative minimal amyloid-forming segment of Amylin, residues 22-27, also form amyloid when Phe-23 is replaced by Leu. Amyloid formation is more facile when the N terminus is deprotonated and the peptide is uncharged. Substitution of the Phe with Ala results in a peptide that is noticeably less prone to form amyloid. A peptide corresponding to residues 10-19 of human Amylin with blocked termini and the sole aromatic residue, Phe-15, substituted by Leu readily forms amyloid. A Phe-15 to Ala substitution reduces significantly the ability to form amyloid. These results indicate that an aromatic residue is not required for amyloid formation in these systems and indicates that other factors such as size, beta-sheet propensity, and hydrophobicity of the side chain in question are also important. 相似文献
5.
Roles of the exposed aromatic residues in crystalline chitin hydrolysis by chitinase A from Serratia marcescens 2170 总被引:1,自引:0,他引:1
Uchiyama T Katouno F Nikaidou N Nonaka T Sugiyama J Watanabe T 《The Journal of biological chemistry》2001,276(44):41343-41349
Four exposed aromatic residues, two in the N-terminal domain (Trp-69 and Trp-33) and two in the catalytic domain (Trp-245 and Phe-232) of Serratia marcescens chitinase A, are linearly aligned with the deep catalytic cleft. To investigate the importance of these residues in the binding activity and hydrolyzing activity against insoluble chitin, site-directed mutagenesis to alanine was carried out. The substitution of Trp-69, Trp-33, or Trp-245 significantly reduced the binding activity to both highly crystalline beta-chitin and colloidal chitin. The substitution of Phe-232, which is located closest to the catalytic cleft, did not affect the binding activity. On the other hand, the hydrolyzing activity against beta-chitin microfibrils was significantly reduced by the substitution of any one of the four aromatic residues including Phe-232. None of the mutations reduced the hydrolyzing activity against soluble substrates. These results clearly demonstrate that the four exposed aromatic residues are essential determinants for crystalline chitin hydrolysis. Three of them, two in the N-terminal domain and one in the catalytic domain, play vital roles in the chitin binding. Phe-232 appeared to be important for guiding the chitin chain into the catalytic cleft. Based on these observations, a model for processive hydrolysis of crystalline chitin by chitinase A is proposed. 相似文献
6.
7.
Putative helix F contributes to regioselectivity of hydroxylation in mitochondrial cytochrome P450 27A1. 总被引:2,自引:0,他引:2
On the basis of alignment with structurally characterized cytochromes P450 (P450s), we have identified the putative F and G helices of mitochondrial P450s 27A1 and 11A. We introduced substitutions at Phe-207, Ile-211, and Phe-215 within putative helix F and at Trp-235 and Tyr-238 within putative helix G in P450 27A1 and compared wild type and mutants with respect to catalytic activity, product pattern, substrate binding, formation of hydrogen peroxide, and interaction with redox partner. Results indicate that the mutated residues are important for delivery of the correctly oriented substrate to the P450 active site. The I211K and F215K mutations, for example, affected the regioselectivity of P450 27A1-dependent hydroxylation reactions and conferred the P450 capacity to cleave the C-C bond of the substrate during the catalytic cycle. Studies of P450 11A1 indicate that Phe-202 has functions similar to those of its counterpart in P450 27A1 (Phe-215). We propose that putative helices F and G form the sides of the substrate-access channel, thus providing the additional mechanism to control regioselectivity of hydroxylation in mitochondrial P450s. 相似文献
8.
Zhao Y Sun L Muralidhara BK Kumar S White MA Stout CD Halpert JR 《Biochemistry》2007,46(41):11559-11567
The crystal structure of P450 2B4 bound with 1-(4-chlorophenyl)imidazole (1-CPI) has been determined to delineate the structural basis for the observed differences in binding affinity and thermodynamics relative to 4-(4-chlorophenyl)imidazole (4-CPI). Compared with the previously reported 4-CPI complex, there is a shift in the 1-CPI complex of the protein backbone in helices F and I, repositioning the side chains of Phe-206, Phe-297, and Glu-301, and leading to significant reshaping of the active site. Phe-206 and Phe-297 exchange positions, with Phe-206 becoming a ligand-contact residue, while Glu-301, rather than hydrogen bonding to the ligand, flips away from the active site and interacts with His-172. As a result the active site volume expands from 200 A3 in the 4-CPI complex to 280 A3 in the 1-CPI complex. Based on the two structures, it was predicted that a Phe-206-->Ala substitution would alter 1-CPI but not 4-CPI binding. Isothermal titration calorimetry experiments indicated that this substitution had no effect on the thermodynamic signature of 4-CPI binding to 2B4. In contrast, relative to wild-type 1-CPI binding to F206A showed significantly less favorable entropy but more favorable enthalpy. This result is consistent with loss of the aromatic side chain and possible ordering of water molecules, now able to interact with Glu-301 and exposed residues in the I-helix. Hence, thermodynamic measurements support the active site rearrangement observed in the crystal structure of the 1-CPI complex and illustrate the malleability of the active site with the fine-tuning of residue orientations and thermodynamic signatures. 相似文献
9.
Hitoshi Ueda Hiromi Iyo Mitsunobu Doi Masatoshi Inoue Toshimasa Ishida Hiroshi Morioka Toshiki Tanaka Satoshi Nishikawa Seiichi Uesugi 《FEBS letters》1991,280(2):207-210
Four mutants of the human cap binding protein (hCBP), in which Trp-102, Glu-103, Asp-104 or Glu-105 was changed to the aliphatic Leu or Ala, were prepared, and their cap binding abilities were examined. Cap binding abilities of two mutants. W102L (Trp-102→Leu) and E105A (Glu-105→Ala), were significantly decreased in comparison with the wild-type hCBP. This result suggest that Trp-102 and Glu-105 are both necessary for the cap binding, and the most probable binding mode with the m7G of cap structure is the combination of the stacking by Trp-102 and the hydrogen-bond pairing by Glu-105, as was already proposed from the model studies. 相似文献
10.
Three mutant Escherichia coli aspartate aminotransferases in which Val39 was changed to Ala, Leu, and Phe by site-directed mutagenesis were prepared and characterized. Among the three mutant and the wild-type enzymes, the Leu39 enzyme had the lowest Km values for dicarboxylic substrates. The Km values of the Ala39 enzyme for dicarboxylates were essentially the same as those of the wild-type (Val39) enzyme. These two mutant enzymes showed essentially the same kcat values for dicarboxylic substrates as did the wild-type enzyme. On the other hand, incorporation of a bulky side-chain at position 39 (Phe39 enzyme) decreased both the affinity (1/Km) and catalytic ability (kcat) toward dicarboxylic substrates. These results show that the position 39 residue is involved in the modulation of both the binding of dicarboxylic substrates to enzyme and the catalytic ability of the enzyme. Although the replacement of Val39 with other residues altered both the kcat and Km values toward various substrates including dicarboxylic and aromatic amino acids and the corresponding oxo acids, it did not alter the ratio of the kcat/Km value of the enzyme toward a dicarboxylic substrate to that for an aromatic substrate. The affinity for aromatic substrates was not affected by changing the residue at position 39. These data indicate that, although the side chain bulkiness of the residue at position 39 correlates well with the activity toward aromatic substrates in the sequence alignment of several aminotransferases [Seville, M., Vincent M.G., & Hahn, K. (1988) Biochemistry 27, 8344-8349], the residue does not seem to be involved in the recognition of aromatic substrates. 相似文献
11.
B Vilsen 《FEBS letters》1992,314(3):301-307
Site-specific mutagenesis was used to analyse the functional roles of the residues Pro328 and Leu332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the alpha 1-subunit of the ouabain resistant rat kidney Na+,K(+)-ATPase. cDNAs encoding either of the Na+,K(+)-ATPase mutants Pro328-->Ala and Leu332-->Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS-1 cells. Ouabain-resistant clones growing in the presence of 10 microM ouabain were isolated, and the Na+,K+, ATP and pH dependencies of the Na+,K(+)-ATPase activity measured in the presence of 10 microM ouabain were analysed. Under these conditions the exogenous expressed Na+,K(+)-ATPase contributed more than 95% of the Na+,K(+)-ATPase activity. The Pro328-->Ala mutant displayed a reduced apparent affinity for Na+ (K0.5 (Na+) 13.04 mM), relative to the wild type (K0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu332-->Ala mutant was increased (K0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K0.5 (K+) 2.46 mM for Pro328-->Ala and 1.97 mM for Leu332-->Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type (K0.5 (ATP) 0.086 mM for Pro328-->Ala and 0.042 mM for Leu332-->Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)-->E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro328 and Leu332 in the stabilization of the E2 form and of Pro328 in Na+ binding. The possible role of the mutated residues in K+ binding is discussed. 相似文献
12.
Jose R. Casas-Finet Mustafa I. Khamis August H. Maki John W. Chase 《FEBS letters》1987,220(2):347-352
The binding of both wild-type and point-mutated E. coli single-stranded DNA-binding (SSB) protein to poly(deoxythymidylic acid) has been studied by fluorescence and optical detection of triplet state magnetic resonance spectroscopy. Involvement of tryptophan residues 40 and 54 in stacking interactions with nucleotide bases has been inferred earlier from such studies. Investigation of a point mutation in the E. coli SSB gene product obtained by site specific oligonucleotide mutagenesis in which Phe-60 is replaced by alanine strongly suggests the participation of Phe-60 in the binding process, possibly by the formation of an extended stacking structure by Trp-54, thymine and Phe-60. This hypothesis is supported by results on the point mutations in which His-55 is replaced by either leucine or tyrosine. 相似文献
13.
El-Mashtoly SF Nakashima S Tanaka A Shimizu T Kitagawa T 《The Journal of biological chemistry》2008,283(27):19000-19010
The direct oxygen sensor protein isolated from Escherichia coli (Ec DOS) is a heme-based signal transducer protein responsible for phosphodiesterase (PDE) activity. Binding of O(2), CO, or NO to a reduced heme significantly enhances the PDE activity toward 3',5'-cyclic diguanylic acid. We report stationary and time-resolved resonance Raman spectra of the wild-type and several mutants (Glu-93 --> Ile, Met-95 --> Ala, Arg-97 --> Ile, Arg-97 --> Ala, Arg-97 --> Glu, Phe-113 --> Leu, and Phe-113 --> Thr) of the heme-containing PAS domain of Ec DOS. For the CO- and NO-bound forms, both the hydrogen-bonded and non-hydrogen-bonded conformations were found, and in the former Arg-97 forms a hydrogen bond with the heme-bound external ligand. The resonance Raman results revealed significant interactions of Arg-97 and Phe-113 with a ligand bound to the sixth coordination site of the heme and profound structural changes in the heme propionates upon dissociation of CO. Mutation of Phe-113 perturbed the PDE activities, and the mutation of Arg-97 and Phe-113 significantly influenced the transient binding of Met-95 to the heme upon photodissociation of CO. This suggests that the electrostatic interaction of Arg-97 and steric interaction of Phe-113 are crucial for regulating the competitive recombination of Met-95 and CO to the heme. On the basis of these results, we propose a model for the role of the heme propionates in communicating the heme structural changes to the protein moiety. 相似文献
14.
Ligand migration in human myoglobin: steric effects of isoleucine 107(G8) on O(2) and CO binding
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To investigate the ligand pathway in myoglobin, some mutant myoglobins, in which one of the amino acid residues constituting a putative ligand-docking site, Ile107, is replaced by Ala, Val, Leu, or Phe, were prepared and their structural and ligand binding properties were characterized. The kinetic barrier for the ligand entry to protein inside was lowered by decreasing the side-chain volume at position 107, indicating that the bulky side chain interferes with the formation of the activation state for the ligand migration and the free space near position 107 would be filled with the ligand in the activation state. Another prominent effect of the reduced side-chain volume at position 107 is to stabilize the ligand-binding intermediate state. Because the stabilization can be ascribed to decrease of the positive enthalpy, the enlarged free space near position 107 would relieve unfavorable steric interactions between the ligand and nearby amino acid residues. The side-chain volume at position 107, therefore, is crucial for the kinetic barrier for the ligand migration and free energy of the ligand-binding intermediate state, which allows us to propose that some photodissociated O(2) moves toward position 107 to be trapped and then expelled to the solvent. 相似文献
15.
Yun YS Lee TH Nam GH Jang DS Shin S Oh BH Choi KY 《The Journal of biological chemistry》2003,278(30):28229-28236
Two homologous Delta5-3-ketosteroid isomerases from Comamonas testosteroni (TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH activity profiles. TI-WT loses activity below pH 5.0 due to the protonation of the conserved catalytic base, Asp-38, while PI-WT does not. Based on the structural analysis of PI-WT, the critical catalytic base, Asp-38, was found to form a hydrogen bond with the indole ring NH of Trp-116, which is homologously replaced with Phe-116 in TI-WT. To investigate the role of Trp-116, we prepared the F116W mutant of TI-WT (TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared kinetic parameters of those mutants at different pH levels. PI-W116F exhibited significantly decreased catalytic activity at acidic pH like TI-WT, whereas TI-F116W maintained catalytic activity at acidic pH like PI-WT and increased the kcat/Km value by 2.5- to 4.7-fold compared with TI-WT at pH 3.8. The crystal structure of TI-F116W clearly showed that the indole ring NH of Trp-116 could form a hydrogen bond with the carboxyl oxygen of Asp-38 like that of PI-WT. The present results demonstrate that the activities of both PI-WT and TI-F116W at low pH were maintained by a tryptophan, which was able not only to lower the pKa value of the catalytic base but also to increase the substrate affinity. This is one example of the strategy nature can adopt to evolve the diversity of the catalytic function in the enzymes. Our results provide insight into deciphering the molecular evolution of the enzyme and creating novel enzymes by protein engineering. 相似文献
16.
Malherbe P Kratochwil N Knoflach F Zenner MT Kew JN Kratzeisen C Maerki HP Adam G Mutel V 《The Journal of biological chemistry》2003,278(10):8340-8347
A model of the rmGlu1 seven-transmembrane domain complexed with a negative allosteric modulator, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)- 1,6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC) was constructed. Although the mGlu receptors belong to the family 3 G-protein-coupled receptors with a low primary sequence similarity to rhodopsin-like receptors, the high resolution crystal structure of rhodopsin was successfully applied as a template in this model and used to select residues for site-directed mutagenesis. Three mutations, F801(6.51)A, Y805(6.55)A, and T815(7.39)M caused complete loss of the [(3)H]EM-TBPC binding and blocked the EM-TBPC-mediated inhibition of glutamate-evoked G-protein-coupled inwardly rectifying K(+) channel current and [Ca(2+)](i) response. The mutation W798(6.48)F increased the binding affinity of antagonist by 10-fold and also resulted in a marked decrease in the IC(50) value (4 versus 128 nm) compared with wild type. The V757(5.47)L mutation led to a dramatic reduction in binding affinity by 13-fold and a large increase in the IC(50) value (1160 versus 128 nm). Two mutations, N7474(5.51)A and N7504(5.54)A, increased the efficacy of the EM-TBPC block of the glutamate-evoked [Ca(2+)](i) response. We observed a striking conservation in the position of critical residues. The residues Val-757(5.47), Trp-798(6.48), Phe-801(6.51), Tyr-805(6.55), and Thr-815(7.39) are critical determinants of the EM-TBPC-binding pocket of the mGlu1 receptor, validating the rhodopsin crystal structure as a template for the family 3 G-protein-coupled receptors. In our model, the aromatic ring of EM-TBPC might interact with the cluster of aromatic residues formed from Trp-798(6.48), Phe-801(6.51), and Tyr-805(6.55), thereby blocking the movement of the TM6 helix, which is crucial for receptor activation. 相似文献
17.
Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyzes the decarboxylation of l-Orn to putrescine, a rate-limiting step in the formation of polyamines. The X-ray crystal structures of ODC, complexed to several ligands, support a model where the substrate is oriented with the carboxyl-leaving group buried on the re face of the PLP cofactor. This binding site is composed of hydrophobic and electron-rich residues, in which Phe-397 is predicted to form a close contact. Mutation of Phe-397 to Ala reduces the steady-state rate of product formation by 150-fold. Moreover, single turnover analysis demonstrates that the rate of the decarboxylation step is decreased by 2100-fold, causing this step to replace product release as the rate-limiting step in the mutant enzyme. These data support the structural prediction that the carboxyl-leaving group is positioned to interact with Phe-397. Multiwavelength stopped-flow analysis of reaction intermediates suggests that a major product of the reaction with the mutant enzyme is pyridoximine 5'-phosphate (PMP), resulting from incorrect protonation of the decarboxylated intermediate at the C4' position. This finding was confirmed by HPLC analysis of the reaction products, demonstrating that Phe-397 also plays a role in maintaining the integrity of the reaction chemistry. The finding that the carboxylate-leaving group is oriented on the buried side of the PLP cofactor suggests that ODC facilitates decarboxylation by destabilizing the charged substrate carboxyl group in favor of an electrostatically more neutral transition state. 相似文献
18.
Linderoth NA Simon MN Hainfeld JF Sastry S 《The Journal of biological chemistry》2001,276(14):11049-11054
Vaccination with heat shock protein gp96-antigenic peptide complexes produces a powerful specific immune response against cancers and infectious diseases in some experimental animal models, and gp96-peptide complexes are now being tested in human clinical trials. gp96 appears to serve as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways. A fundamental issue that needs to be addressed is the mechanism of binding of antigenic peptide to gp96. Here, we show using scanning transmission electron microscopy that recombinant gp96 binds peptide in stable multimeric complexes, which may have biological significance. To open the possibility for genetically engineering gp96 for improved immunogenicity and to understand if molecular recognition plays a role in the binding of antigenic peptide, we mutagenized some specific aromatic amino acids in the presumed peptide-binding pocket. Replacement of Tyr-667 or Tyr-678 to Ala reduced affinity for peptide whereas conversion of Trp-654 to Tyr increased peptide binding. Similarly, changing Trp-621 to Phe or Leu or Ala or Ile negatively affected peptide binding whereas changing Trp-621 to Tyr or Val positively affected peptide binding. Probing the peptide microenvironment in gp96-peptide complexes, suggested that hydrophobic interactions (and perhaps hydrogen bonding/stacking interactions) may play a role in peptide loading by gp96. 相似文献
19.
K D Ridge S Bhattacharya T A Nakayama H G Khorana 《The Journal of biological chemistry》1992,267(10):6770-6775
In order to prepare a completely light-stable rhodopsin, we have synthesized an analog, II, of 11-cis retinal in which isomerization at the C11-C12 cis-double bond is blocked by formation of a cyclohexene ring from the C10 to C13-methyl. We used this analog to generate a rhodopsin-like pigment from opsin expressed in COS-1 cells and opsin from rod outer segments (Bhattacharya, S., Ridge, K.D., Knox, B.E., and Khorana, H. G. (1992) J. Biol. Chem. 267, 6763-6769). The pigment (lambda max, 512 nm) formed from opsin and analog II (rhodospin-II) showed ground state properties very similar to those of rhodopsin, but was not entirely stable to light. In the present work, 12 opsin mutants (Ala-117----Phe, Glu-122----Gln(Ala, Asp), Trp-126----Phe(Leu, Ala), Trp-265----Ala(Tyr, Phe), Tyr-268----Phe, and Ala-292----Asp), where the mutations were presumed to be in the retinal binding pocket, were reconstituted with analog II. While all mutants formed rhodopsin-like pigments with II, blue-shifted (12-30 nm) chromophores were obtained with Ala-117----Phe, Glu-122----Gln(Ala), Trp-126----Leu(Ala), and Trp-265----Ala(Tyr, Phe) opsins. The extent of chromophore formation was markedly reduced in the mutants Ala-117----Phe and Trp-126----Ala. Upon illumination, the reconstituted pigments showed varying degrees of light sensitivity; the mutants Trp-126----Phe(Leu) showed light sensitivity similar to wild-type. Continuous illumination of the mutants Glu-122----Asp, Trp-265----Ala, Tyr-268----Phe, and Ala-292----Asp resulted in hydrolysis of the retinyl Schiff base. Markedly reduced light sensitivity was observed with the mutant Trp-265----Tyr, while the mutant Trp-265----Phe was light-insensitive. Consistent with this result, the mutant Trp-265----Phe showed no detectable light-dependent activation of transducin or phosphorylation by rhodopsin kinase. 相似文献
20.
Clausen JD McIntosh DB Vilsen B Woolley DG Andersen JP 《The Journal of biological chemistry》2003,278(22):20245-20258
Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein. 相似文献