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1.
Site-directed mutagenesis and X-ray absorption spectroscopy studies have previously shown that the tRNA-guanine transglycosylase (TGT) from Escherichia coli is a zinc metalloprotein and identified the enzymic ligands to the zinc [Chong et al. (1995), Biochemistry
34, 3694–3701; Garcia et al. (1966), Biochemistry
35, 3133–3139]. During these studies one mutant, TGT (C265A), was found to exhibit a significantly lower specific activity, but was not found to be involved in the zinc site. The present report demonstrates that TGT is inactivated by treatment with thiol reagents (e.g., DTNB, MMTS, and N-ethylmaleimide). Further, this inactivation is shown to be due to modification of cysteine 265. The kinetic parameters for the mutants TGT (C265A) and TGT (C265S), however, suggest that this residue is not performing a critical role in the TGT reaction. We conclude that cysteine 265 is in the active site of TGT, but is not performing a critical catalytic function. This conclusion is supported by the recent determination of the X-ray crystal structure of the TGT from Zymomonas mobilis [Romier et al. (1966), EMBO J.
15, 2850–2857], which reveals that the residue corresponding to cysteine 265 is distant from the putative catalytic site, but is in the middle of a region of the enzyme surface proposed to bind tRNA. 相似文献
2.
N. Yu. Glazova E. A. Sebentsova N. G. Levitskaya L. A. Andreeva L. Yu. Alfeeva A. A. Kamenskii N. F. Myasoedov 《Biology Bulletin》2005,32(4):381-386
A comparative study of nootropic activity of semax (MEHFPGP), an analog of ACTH4–10, and some of its derivatives, in which the N-terminal methionine was modified or substituted with other amino acid residues, was performed. The effect of these peptides on learning of albino rats in tests with positive (food) and negative (pain) reinforcement was studied. In the case of modification of methionine by attachment of the gluconic-acid residue or substitution of methionine with lysine, the nootropic effect of the peptide was retained. The substitution of methionine with tryptophan or serine resulted in a decrease in the nootropic activity. The substitution of methionine with glycine, threonine, or alanine caused a complete loss of the nootropic activity of the peptide. Therefore, the amino acid residue located at position 1 of the heptapeptide analog semax, plays a key role in retaining the nootropic effects of the peptide and determines the degree of their expression.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 4, 2005, pp. 460–466.Original Russian Text Copyright © 2005 by Glazova, Sebentsova, Levitskaya, Andreeva, Alfeeva, Kamenskii, Myasoedov. 相似文献
3.
Jerrolynn D. Hockenhull-Johnson Mary S. Stern Jonathan B. Wittenberg Serge N. Vinogradov Oscar H. Kapp Daniel A. Walz 《Journal of Protein Chemistry》1993,12(3):261-277
The cytoplasmic hemoglobin III from the gill of the symbiont-harboring clamLucina pectinata consists of 152 amino acid residues, has a calculated Mm of 18,068, including heme, and has N-acetyl-serine as the N-terminal residue. Based on the alignment of its sequence with other vertebrate and nonvertebrate globins, it retains the invariant residues Phe45 at position CD1 and His98 at the proximal position F8, as well as the highly conserved Trp16 and Pro39 at positions A12 and C2, respectively. The most likely candidate for the distal residue at position E7 is Gln66.Lucina hemoglobin III shares 95 identical residues with hemoglobin II (J. D. Hockenhull-Johnsonet al., J. Prot. Chem.
10, 609–622, 1991), including Tyr at position B10, which has been shown to be capable of entering the distal heme cavity and placing its hydroxyl group within a 2.8 Å of the water molecule occupying the distal ligand position, by modeling the hemoglobin II sequence using the crystal structure of sperm whale metmyoglobin. The amino acid sequences of the twoLucina globins are compared in detail with the known sequences of mollusc globins, including seven cytoplasmic and 11 intracellular globins. Relative to 75% homology between the twoLucina globins (counting identical and conserved residues), both sequences have percent homology scores ranging from 36–49% when compared to the two groups of mollusc globins. The highest homology appears to exist between theLucina globins and the cytoplasmic hemoglobin ofBusycon canaliculatum. 相似文献
4.
Beier JI Kaiser JP Guo L Martínez-Maldonado M Arteel GE 《Archives of biochemistry and biophysics》2011,(1):19-26
Fungal methionine synthase, Met6p, transfers a methyl group from 5-methyl-tetrahydrofolate to homocysteine to generate methionine. The enzyme is essential to fungal growth and is a potential anti-fungal drug design target. We have characterized the enzyme from the pathogen Candida albicans but were unable to crystallize it in native form. We converted Lys103, Lys104, and Glu107 all to Tyr (Met6pY), Thr (Met6pT) and Ala (Met6pA). All variants showed wild-type kinetic activity and formed useful crystals, each with unique crystal packing. In each case the mutated residues participated in beneficial crystal contacts. We have solved the three structures at 2.0–2.8 Å resolution and analyzed crystal packing, active-site residues, and similarity to other known methionine synthase structures. C. albicans Met6p has a two domain structure with each of the domains having a (βα)8-barrel fold. The barrels are arranged face-to-face and the active site is located in a cleft between the two domains. Met6p utilizes a zinc ion for catalysis that is bound in the C-terminal domain and ligated by four conserved residues: His657, Cys659, Glu679 and Cys739. 相似文献
5.
Konrad L. Maier Eva Matejkova Helga Hinze Lieselotte Leuschel Hans Weber Ingrid Beck-Speier 《FEBS letters》1989,250(2)
Oxidation of the reactive site methionine (Met) in α-1-proteinase inhibitor (α-1-PI) to methionine sulfoxide (Met(O)) is known to cause depletion of its elastase inhibitory activity. To estimate the selectivity of different oxidants in converting Met to Met(O) in α-1-PI, we measured the molar ratio Met(O)/α-1-PI at total inactivation. This ratio was determined to be 1.2 for both the myeloperoxidase/H2O2/chloride system and the related compound NH2Cl. With taurine monochloramine, another myeloperoxidase-related oxidant, 1.05 mol Met(O) were generated per mol α-1-PI during inactivation. These oxidants attack preferentially one Met residue in α-1-PI, which is identical with Met 358, as concluded from the parallelism of loss of elastase inhibitory activity and oxidation of Met. A similar high specificity for Met oxidation was determined for the xanthine oxidase-derived oxidants. In contrast, the ratio found for ozone and m-chloroperoxybenzoic acid was 6.0 and 5.0, respectively, indicating oxidation of additional Met residues besides the reactive site Met in α-1-PI, i.e. unselective action of these oxidants. Further studies were performed on the efficiency of oxidants for total depletion of the elastase inhibitory capacity of α-1-PI. Ozone and m-chloroperoxybenzoic acid were 10-fold less effective and the superoxide anion/hydroxyl radicals were 30–50-fold less effective to inactivate the elastase inhibitory activity as compared to the myeloperoxidase-derived oxidants. The myeloperoxidase-related oxidants are discussed as important regulators of α-1-PI activity in vivo. 相似文献
6.
Joye IJ Beliën T Brijs K Soetaert W Delcour JA 《Applied microbiology and biotechnology》2011,90(1):173-180
Nucleotide pyrophosphatases/phosphodiesterases (NPPs, PF01663) release nucleoside 5′-monophosphates from a wide range of nucleotide
substrates. Only very recently, the first plant members of the NPP family were characterised (Joye et al. J Cereal Sci 51:
326–336, 2010), and little is known about their substrate-specifying residues. We elucidated the role of six amino acid residues of the
recently identified and characterised Triticum aestivum L. NPP (Joye et al. J Cereal Sci 51: 326–336, 2010). Substitution of the highly conserved catalytic Thr132 into Ser or Ala completely abolished enzyme activity. Mutation of
a highly conserved His255 residue into an apolar Ala suprisingly increased enzyme activity against most phosphodiester substrates.
Four other residues moderately to highly conserved over NPPs of different organisms were studied as well. Mutation of the
Asn153, Asn165 and Glu199 into an Arg, Ser and Asp residue, respectively, increased the relative enzyme activity against p-nitrophenyl phosphate. Furthermore, mutation of Phe194 into Ser increased the relative enzyme activity against adenosine 5′-monophosphate-containing
substrates, although the overall enzyme activity of this mutant enzyme decreased. We conclude that the structural requirements
and the conservation of the amino acids of the catalytic site of TaNPPr and, by extension, probably of all NPPs, are very
stringent. 相似文献
7.
Jasmin Mecinovi Rasheduzzaman Chowdhury Emily Flashman Christopher J. Schofield 《Analytical biochemistry》2009,393(2):215-221
Prolyl hydroxylase domain 2 (PHD2) plays an important role in hypoxic sensing in humans. Here we report studies on the reactivity of cysteinyl residues of the catalytic domain of PHD2 using an approach in which nondenaturing electrospray ionization–mass spectrometry (ESI–MS) analyses were combined with the use of a thiol library and residue substitution. Among the seven cysteinyl residues of the PHD2 catalytic domain, Cys201 was found to be predominantly modified by thiols or N-ethylmaleimide. Selective modification of Cys201 was further demonstrated with methanethiosulfonate, a spin-labeled probe. The modified PHD2 will be useful in electron paramagnetic resonance studies on PHD2. The results demonstrate the use of a combined library/residue substitution/ESI–MS approach for analyzing residue reactivity. 相似文献
8.
Nabil G. Seidah James A. Cromlish Josée Hamelin Gaétan Thibault Michel Chrétien 《Bioscience reports》1986,6(9):835-844
IRCM-Serine Protease 1 (IRCM-SP1) has recently been isolated and characterized from porcine pituitary anterior and neurointermediate lobes (Cromlishet al., 1986a,J. Biol. Chem.
261:10850–10858; Cromlishet al., 1986b,J. Biol. Chem.
261:10859–10870). This pituitary serine protease was shown to selectively cleave human proopiomelanocortin (POMC)-derived peptides at both pairs of basic residues and C-terminal to specific Arg residues, all known to be cleavedin vivo. Here, a similar enzyme was isolated from rat heart atria and ventricles. Rat IRCM-SP1 was shown to be highly specific for the same cleavage sites in POMC, as the porcine pituitary homologue. Furthermore, the rat and the porcine enzymes cleave rat pro-Atrial Natriuretic Factor (pro-ANF 1–126) to yield ANF 103–126, 102–126 and 99–126 in that order of preference. This suggests thatin vitro the cleavage sites preferred in pro-ANF resemble those found in brain and hypothalamus. The enzyme is nine times more abundant in atria versus ventricles/mg protein. It is concluded that IRCM-SP1, could well represent a common pro-hormone maturation enzyme for POMC and Pro-ANF and possibly many other pro-hormones. 相似文献
9.
Botulinum neurotoxin (NT) serotype A is a 150-kDa dichain protein. Posttranslational nicking of the single-chain NT (residues Pro 1–Leu 1295) by the protease(s) endogenous to Clostridium botulinum excises 10 residues, leaving Pro 1–Lys 437 and Ala 448–Leu 1295 in the 50-kDa light (L) and 100-kDa heavy (H) chains, respectively, connected by a Cys 429–Cys 453 disulfide and noncovalent bonds [Krieglstein et al. (1994), J. Protein Chem.
13, 49–57]. The L chain is a metalloprotease, while the amino- and carboxy-terminal halves of the H chain have channel-forming and receptor-binding activities, respectively [Montecucco and Schiavo (1995), Q. Rev. Biophys.
28, 423–472]. Endoproteinase Glu-C and -chymotrypsin were used for controlled digestion at pH 7.4 of the 150-kDa dichain NT and the isolated 100-kDa H chain (i.e., freed from the L chain) in order to map the cleavage sites and isolate the proteolytic fragments. The dichain NT appeared more resistant to cleavage by endoproteinase Glu-C than the isolated H chain. In contrast, the NT with its disulfide(s) reduced showed rapid digestion of both chains, including a cleavage between Glu 251 and Met 252 (resulting in 30- and 20-kDa fragments of the L chain) which was not noted unless the NT was reduced. Interestingly, an adjacent bond, Tyr 249–Tyr 250, was noted earlier [DasGupta and Foley (1989), Biochimie
71, 1193–1200] to undergo self-cleavage following reductive separation of the L chain from the H chain. The site Tyr–Tyr–Glu–Met (residues 249–252) appears to become exposed following reduction of Cys 429–Cys 453 disulfide. Identification of Glu 669–Ile 670 and Tyr 683–Ile 684 as protease-susceptible sites demonstrated for the first time that at least two peptide bonds in the segment of the H chain (residues 659–684), part of which (residues 659–681) is thought to interact with the endosomal membranes and forms channels [Oblatt-Montal et al., (1995), Protein Sci.
4, 1490–1497], are exposed on the surface of the NT. Two of the fragments of the H chain we generated and purified by chromatography are suitable for structure–function studies; the 85- and 45-kDa fragments beginning at residue Leu 544 and Ser 884, respectively (both extend presumably to Leu 1295) contain the channel-forming segment and receptor-binding segments, respectively. In determining partial amino acid sequences of 10 fragments, a total of 149 amino acids in the 1275-residue NT were chemically identified. 相似文献
10.
David W. Deerfield II Amanda M. Holland-Minkley Joe Geigel Hugh B. Nicholas Jr. 《Journal of Protein Chemistry》1997,16(5):441-447
We have studied the classification of the environment of residues within protein structures. Eisenberg's original idea created environmental categories to discriminate between similar residues [Bowie et al., Science (1991), 253, 164–170]. These environments grouped residues based upon their buried surface area, polarity of the surrounding environment, and secondary structure element in which the residue is found. However, Eisenberg's original categories led to incomplete discrimination between residues that only partially substitute for each other. We have expanded on Eisenberg's original idea of environmental categories, by both considering additional contacts in the calculation of the solvent-accessible molecular surface area and by subdividing the environmental plot into regions based upon its theoretical features. Our alternative surface area calculations were used in conjunction with the polarity of the environment of the residue to define a new set of environmental categories. These new categories were able to discriminate between residues such as threonine, valine, and aspartic acid while reflecting the propensity of these residues to substitute for each other. 相似文献
11.
Characterization and sequencing of cDNA clone encoding the phloem protein PP2 of Cucurbita pepo 总被引:3,自引:0,他引:3
Direct N-terminal amino acid sequencing of the phloem protein 2 (PP2) from 3-month old Cucurbita pepo L. (pumpkin), purified by SDS-PAGE and blotted onto PVDF membrane, showed that the protein had a blocked N-terminus. However, after in situ cleavage of the polypeptide in a gel slice by cyanogen bromide, 75 residues of sequence on two cyanogen bromide fragments were determined. An oligonucle-otide probe based on this amino acid sequence was used to screen a cDNA library, constructed from mRNA of 3–5-day old seedling hypocotyls, in ZAP II. A cDNA clone (p11A) predicted an amino acid sequence of 218 residues, in full agreement with the sequences determined for two CNBr fragments of PP2, and suggests that the N-terminus of the protein is a blocked methionine residue which is cleaved off by CNBr. Two additional cDNA clones were sequenced but no heterogeneity in the PP2 sequence was found. The deduced amino acid sequence of C. pepo differs in nine residues from the recently published sequence of Cucurbita maxima (Bostwick et al., Plant Cell 4 (1992) 1539–1548). Southern blot showed that PP2 is encoded by a gene family with a relatively large number of members (estimated as 7–15 per haploid genome). 相似文献
12.
Botulinum neurotoxin (NT) serotype E is synthesized by Clostridium botulinum as an 150-kDa single-chain polypeptide of 1252 amino acid residues of which 8 are Cys residues [Puolet et al. (1992), Biochem. Biophys. Res. Commun.
183, 107–113]. The posttranslational processing of the gene product removes only the initiating methionine. A very narrow segment of this 1251-residue-long mature protein—at one-third the distance from the N-terminus (between residues Lys 418 and Arg 421)—is highly sensitive to proteases, such as trypsin. The single-chain NT easily undergoes an exogenous posttranslational modification by trypsin; residues 419–421 (Gly–Ile–Arg) are excised. The proteolytically processed NT is a dichain protein in which Pro 1–Lys 418 constitute the 50–kDa light chain, Lys 422–Lys 1251 constitute the 100–kDa heavy chain; Cys 411–Cys 425 and Cys 1196–Cys 1237 form the interchain and intrachain disulfide bonds, respectively; the other four Cys residues at positions 25, 346, 941, and 1035 remain as free sulfhydryl groups. The 150–kDa dichain NT, and separated light and heavy chains, were fragmented with CNBr and endoproteases (pepsin and clostripain); some of these fragments were carboxymethylated with iodoacetamide (with or without I4C label) before and after fragmentation. The fragments were separated and analyzed for amino acid compositions and sequences by Edman degradation to determine the complete covalent structure of the dichain type E NT. A total of 208 amino acid residues, i.e., 16.5% of the entire protein's sequence deduced from nucleotide sequence, was identified. Direct chemical identification of these amino acids was in complete agreement with that deduced from nucleotide sequence. 相似文献
13.
Grishin NV 《Journal of molecular evolution》1995,41(5):675-679
A general model for estimating the number of amino acid substitutions per site (d) from the fraction of identical residues between two sequences (q) is proposed. The well-known Poisson-correction formula q = e
–d
corresponds to a site-independent and amino-acid-independent substitution rate. Equation q = (1 – e –2d
)/2d, derived for the case of substitution rates that are site-independent, but vary among amino acids, approximates closely the empirical method, suggested by Dayhoff et al. (1978). Equation q = 1/(1 + d) describes the case of substitution rates that are amino acid-independent but vary among sites. Lastly, equation q = [ln(1 + 2d)]/2d accounts for the general case where substitution rates can differ for both amino acids and sites. 相似文献
14.
Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) was completely denatured using 3 M guanidine hydrochloride for 2 h as in previous studies [Yao et al. (1982), Sci. Sin.
25B, 1296–1302; Yao et al. (1984), Biochemistry
23, 2740–2744; Yao et al. (1982), Sci. Sin.
25B, 1186–1193]. Under suitable conditions, about 60–70% of the activity can be recovered in the presence of different Mg2+ concentrations. Both the reactivation and the refolding processes follow two-phase courses after dilution in the proper solutions. A comparison of the rate constants for the refolding of unfolded creatine kinase with those for the recovery of its catalytic activity at various Mg2+ concentrations shows that these are not synchronized. The reactivity of guanidine hydrochloride-denatured creatine kinase can be inhibited by Mg2+; however, the rates of reactivation are independent of the Mg2+ concentration. In addition, Mg2+ affects the fluorescence intensity, but the rate constants of refolding are independent of Mg2+ concentration. Although the reactivation of GdHCl-denatured creatine kinase is complete about 3 h after dilution with reactivation solutions, the conformational changes during refolding occur in a much slower reaction. Mg2+ can induce complex changes in the relative fluorescence intensity during refolding over a broad range of concentrations. 相似文献
15.
Kerstin G. Kriegistein Bibhuti R. DasGupta Agnes H. Henschen 《Journal of Protein Chemistry》1994,13(1):49-57
Botulinum neurotoxin Type A is synthesized byClostridium botulinum as a 150 kD single chain polypeptide. The posttranslational processing of the 1296 amino acid residue long gene product involves removal of the initiating methionine, formation of disulfide bridges, and limited proteolysis (nicking) by the bacterial protease(s). The mature dichain neurotoxin is made of a 50-kD light chain and a 100-kD heavy chain connected by a disulfide bridge. DNA derived amino acid sequencepredicted a total of 9 Cys residues (Binzet al., 1990,J. Biol. Chem.
265, 9153–9158; Thompsonet al., 1990,Eur. J. Biochem.
189, 73–81). Treatment of the dichain neurotoxin, dissolved in 6 M guanidine. HCl, with 4-vinylpyridine converted 5 Cys residues into S-pyridylethyl cysteine residues; but alkylation after mercaptolysis converted all 9 Cys residues in the S-pyridylethylated form. After confirming the predicted number of Cys residues by amino acid analysis, the positions of the 5 Cys residues carrying sulfhydryl groups and the 4 involved in disulfide bridges were determined by comparing the elution patterns in reversed-phase HPLC of the cyanogen bromide mixtures of the exclusively alkylated and the mercaptolyzed-alkylated neurotoxin. The chromatographically isolated components were identified by N-terminal amino acid sequence analysis. The HPLC patterns showed characteristic differences. The Cys residuespredicted in positions 133, 164, 790, 966, and 1059 were found in the sulfhydryl form; Cys 429 and 453 were found disulfide-bridged connecting the light and heavy chains, and Cys 1234 and 1279 were found in an intrachain disulfide-bridge near the C-terminus in the heavy chain. Ten amino acid residues, Thr 438-Lys 447,predicted to be present in the single chain neurotoxin were not found in the dichain neurotoxin. Nicking of single-chain neurotoxin by the protease(s) endogenous to the bacteria therefore appears to excise these 10 amino acid residues from the nicking region which leaves Lys 437 as the C-terminus of the light chain and Ala 448 as the N-terminus of the heavy chain. The N-terminal Pro 1 and C-terminal Leu 1295,predicted from the nucleotide sequence, remain conserved after nicking. Residues Pro 1-Lys 437 and Ala 448-Leu 1295 constitute the light and heavy chains, respectively. The C-termini were determined by isolation of short C-terminal peptide fragments and subsequent sequence analysis by Edman degradation. About 20% of the amino acid sequence predicted from DNA analysis was confirmed in these studies by protein-chemical methods. 相似文献
16.
William Parker Todd A. Wells Susanne Meza-Keuthen In-Soo Kim Pill-Soon Song 《Journal of Protein Chemistry》1995,14(2):53-57
Recently, Mummertet al. [Nature
363, 644–648 (1993)] isolated a proposed TCP1-related chaperone. Here we report several findings concerning the protein which they sequenced. Two similar N-terminal sequences were obtained from this abundant 60-kDa protein. Internal sequences were also acquired by protease digestion. Initially it was believed the protein was able to completely inhibit citrate synthase aggregation, but later purifications demonstrated that the 60-kDa polypeptide lacked both chaperone activity and the previously reported kinase activity [Grimmet al., Planta
178, 199–206 (1989)]. It is now our belief that this protein is neither a chaperone nor a kinase. 相似文献
17.
Eijkelenboom AP van den Ent FM Wechselberger R Plasterk RH Kaptein R Boelens R 《Journal of biomolecular NMR》2000,18(2):119-128
The solution structure of the dimeric N-terminal domain of HIV-2 integrase (residues 1–55, named IN1–55) has been determined using NMR spectroscopy. The structure of the monomer, which was already reported previously [Eijkelenboom et al. (1997) Curr. Biol., 7, 739–746], consists of four -helices and is well defined. Helices 1, 2 and 3 form a three-helix bundle that is stabilized by zinc binding to His12, His16, Cys40 and Cys43. The dimer interface is formed by the N-terminal tail and the first half of helix 3. The orientation of the two monomeric units with respect to each other shows considerable variation. 15N relaxation studies have been used to characterize the nature of the intermonomeric disorder. Comparison of the dimer interface with that of the well-defined dimer interface of HIV-1 IN1–55 shows that the latter is stabilized by additional hydrophobic interactions and a potential salt bridge. Similar interactions cannot be formed in HIV-2 IN1–55 [Cai et al. (1997) Nat. Struct. Biol., 4, 567–577], where the corresponding residues are positively charged and neutral ones. 相似文献
18.
Based on selective labeling by ATP analogues, Lys68 of the Calvin Cycle enzyme phosphoribulokinase (PRK) from spinach has been assigned to the active-site region [Miziorkoet al. (1990),J. Biol. Chem.
265, 3642–3647]. The equivalent position is occupied by lysyl or arginyl residues in the PRK from both prokaryotic and eukaryotic sources, suggesting a requirement for a basic residue at this location. To examine this possibility, we have replaced Lys68 of the spinach enzyme with arginyl, glutaminyl, alanyl, or glutamyl residues by site-directed mutagenesis. All of the mutant enzymes retain substantial kinase activity; and even in the case of the radical substitution by glutamate, theK
m
values for ATP and ribulose 5-phosphate are not perturbed significantly. Glutamate at position-68 may destabilize tertiary structure, because the yield of this mutant protein from transformedE. coli is quite low compared to that of the other proteins in this series. Despite the active-site proximity of Lys68, our results show that this residue does not play a key role in catalysis or substrate binding. 相似文献
19.
Mutant Arg76Gln and Lys290Gln Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases have been prepared and analyzed. No alteration in the apparent kinetic constants were detected for the Arg76Gln mutant enzyme, while the Lys290Gln mutant showed a 12-fold decrease in V
max/K
mADP. These results indicate that Arg76 is not involved in CO2 binding, but support the hypothesis that the binding of this substrate induces a conformational change that protects the region around Arg76 from trypsin action [Herrera et al. (1993) J. Protein Chem.
12, 413–418]. These findings also indicate that Lys290, a highly reactive residue against pyrydoxal phosphate [Bazaes et al. (1995), FEBS Lett.
360, 207–210], does not perform an essential function for the enzyme activity. 相似文献
20.
Paola?Caroppi Federica?Sinibaldi Elisa?Santoni Barry?D.?Howes Laura?Fiorucci Tommaso?Ferri Franca?Ascoli Giulietta?Smulevich Roberto?Santucci
The structural and redox properties of a non-covalent complex reconstituted upon mixing two non-contiguous fragments of horse cytochrome c, the residues 1–38 heme-containing N-fragment with the residues 57–104 C-fragment, have been investigated. With respect to native cyt c, the complex lacks a segment of 18 residues, corresponding, in the native protein, to an omega ()-loop region. The fragment complex shows compact structure, native-like -helix content but a less rigid atomic packing and reduced stability with respect to the native protein. Structural heterogeneity is observed at pH 7.0, involving formation of an axially misligated low-spin species and consequent partial displacement of Met80 from the sixth coordination position of the heme-iron. Spectroscopic data suggest that a lysine (located in the Met80-containing loop, namely Lys72, Lys73, or Lys79) replaces the methionine residue. The residues 1–38/57–104 fragment complex shows an unusual biphasic alkaline titration characterized by a low (pKa1=6.72) and a high pKa-associated state transition (pKa2=8.56); this behavior differs from that of native cyt c, which shows a monophasic alkaline transition (pKa=8.9). The data indicate that the 40s -loop plays an important role in the stability of cyt c and in ensuring a correct alkaline conformational transition of the protein. 相似文献