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1.
Luminous bacteria contain three types of NAD(P)H-FMN oxidoreductases (flavin reductases) with different pyridine nucleotide specificities. Among them, the NADPH-specific flavin reductase from Vibrio harveyi exhibits a uniquely high preference for NADPH. In comparing the substrate specificity, crystal structure, and primary sequence of this flavin reductase with other structurally related proteins, we hypothesize that the conserved Arg203 residue of this reductase is critical to the specific recognition of NADPH. The mutation of this residue to an alanine resulted in only small changes in the binding and reduction potential of the FMN cofactor, the K(m) for the FMN substrate, and the k(cat). In contrast, the K(m) for NADPH was increased 36-fold by such a mutation. The characteristic perturbation of the FMN cofactor absorption spectrum upon NADP(+) binding by the wild-type reductase was abolished by the same mutation. While the k(cat)/K(m,NADPH) was reduced from 1990 x 10(5) to 46 x 10(5) M(-1) min(-1) by the mutation, the mutated variant showed a k(cat)/K(m,NADH) of 4 x 10(5) M(-1) min(-1), closely resembling that of the wild-type reductase. The deuterium isotope effects (D)V and (D)(V/K) for (4R)-[4-(2)H]-NADPH were 1.7 and 1.4, respectively, for the wild-type reductase but were increased to 3.8 and 4.0, respectively, for the mutated variant. Such a finding indicates that the rates of NADPH and NADP(+) dissociation in relation to the isotope-sensitive redox steps were both increased as a result of the mutation. These results all provide support to the critical role of the Arg203 in the specific recognition and binding of NADPH. 相似文献
2.
A direct transfer of the reduced flavin mononucleotide (FMNH(2)) cofactor of Vibrio harveyi NADPH:FMN oxidoreductase (FRP) to luciferase for the coupled bioluminescence reaction has been indicated by recent kinetic studies [Lei, B., and Tu, S.-C. (1998) Biochemistry 37, 14623-14629; Jeffers, C., and Tu, S.-C. (2001) Biochemistry 40, 1749-1754]. For such a mechanism, a complex formation of luciferase with FRP is essential, but until now, no evidence for such a complex has been reported. In this work, FRP was labeled at 1:1 molar ratio with the fluorophore eosin. The labeled enzyme was about 30% active in either the reductase single-enzyme or the luciferase-coupled assay. The labeled FRP in either the holo- or apoenzyme form was similar to the native FRP in undergoing a monomer-dimer equilibrium. By measuring the steady-state fluorescence anisotropy of eosin-labeled FRP, it was shown that luciferase formed a complex at 1:1 molar ratio with the monomer of either the apoenzyme or the holoenzyme form of FRP with K(d) values of 7 and 11 microM, respectively. Neither the holo- nor the apoenzyme of the labeled FRP in the dimeric form was effective in complexing with luciferase. At maximal in vivo bioluminescence, the V. harveyi cellular contents of luciferase and FRP were estimated to be 172 and 3 microM, respectively. The vast majority of FRP would be trapped in the luciferase/FRP complex. Plausible physiological significance of such a finding is discussed. 相似文献
3.
The Vibrio harveyi NADPH-specific flavin reductase FRP follows a ping-pong mechanism but switches to a sequential mechanism in the luciferase-coupled reaction. The bound FMN co-isolated with FRP, while acting as a genuine cofactor in the single-enzyme reaction, functions in the luciferase-coupled reaction as a prebound substrate and is directly transferred to luciferase once it is reduced [Lei, B., and Tu, S.-C. (1998) Biochemistry 37, 14623-14629]. With the aim of better understanding the functions of FMN in the FRP holoenzyme, this study was undertaken to quantify and compare the thermodynamic properties of the binding of oxidized and reduced FMN by the FRP apoenzyme. By isothermal titration calorimetry (ITC) measurements in various buffers at pH 7.0 and 15-30 degrees C, the binding of FMN by apo-FRP was found to be noncooperative, exothermic, and primarily enthalpy driven. The binding free energy change (hence, the association constant) was nearly invariant over this temperature range. Significant conformational changes in FRP upon binding of FMN were indicated. Equilibrium bindings of reduced flavins by flavin-dependent proteins have rarely been studied. In this work, the thermodynamic properties of binding of reduced FMN by apo-FRP were found to closely resemble those of FMN binding under three sets of experimental conditions via ITC measurements and, in one case, fluorescence quenching. The kinetically deduced ping-pong mechanism of FRP is now supported by direct measurements of binding affinities of the oxidized and reduced FMN cofactors. These findings are also discussed in relation to the function of FRP as a reduced flavin donor in the FRP-luciferase couple. 相似文献
4.
Vibrio harveyi NADPH-flavin oxidoreductase: cloning, sequencing and overexpression of the gene and purification and characterization of the cloned enzyme. 总被引:5,自引:3,他引:2
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NAD(P)H-flavin oxidoreductases (flavin reductases) from luminous bacteria catalyze the reduction of flavin by NAD(P)H and are believed to provide the reduced form of flavin mononucleotide (FMN) for luciferase in the bioluminescence reaction. By using an oligonucleotide probe based on the partial N-terminal amino acid sequence of the Vibrio harveyi NADPH-FMN oxidoreductase (flavin reductase P), a recombinant plasmid, pFRP1, was obtained which contained the frp gene encoding this enzyme. The DNA sequence of the frp gene was determined; the deduced amino acid sequence for flavin reductase P consists of 240 amino acid residues with a molecular weight of 26,312. The frp gene was overexpressed, apparently through induction, in Escherichia coli JM109 cells harboring pFRP1. The cloned flavin reductase P was purified to homogeneity by following a new and simple procedure involving FMN-agarose chromatography as a key step. The same chromatography material was also highly effective in concentrating diluted flavin reductase P. The purified enzyme is a monomer and is unusual in having a tightly bound FMN cofactor. Distinct from the free FMN, the bound FMN cofactor showed a diminished A375 peak and a slightly increased 8-nm red-shifted A453 peak and was completely or nearly nonfluorescent. The Kms for FMN and NADPH and the turnover number of this flavin reductase were determined. In comparison with other flavin reductases and homologous proteins, this flavin reductase P shows a number of distinct features with respect to primary sequence, redox center, and/or kinetic mechanism. 相似文献
5.
Relaxation characteristics of the 23Na nuclei magnetization were used to determine the sodium-binding properties of the Na+-translocating NADH:quinone oxidoreductase from Vibrio harveyi (NQR). The dissociation constant of Na+ for the oxidized enzyme was found to be 24 mM and for the reduced enzyme about 30 microM. Such large (3 orders in magnitude) redox dependence of the NQR affinity to sodium ions shows that the molecular machinery was designed to use the drop in redox energy for creating an electrochemical sodium gradient. Redox titration of NQR monitored by changes in line width of the 23Na NMR signal at 2 mM Na+ showed that the enzyme affinity to sodium ions follows the Nernst law for a one-electron carrier with Em about -300 mV (vs SHE). The data indicate that energy conservation by NQR involves a mechanism modulating ion affinity by the redox state of an enzyme redox cofactor. 相似文献
6.
Vibrio harveyi NADPH:FMN oxidoreductase P (FRP(Vh)) is a homodimeric enzyme having a bound FMN per enzyme monomer. The bound FMN functions as a cofactor of FRP(Vh) in transferring reducing equivalents from NADPH to a flavin substrate in the absence of V. harveyi luciferase but as a substrate for FRP(Vh) in the luciferase-coupled bioluminescent reaction. As part of an integral plan to elucidate the regulation of functional coupling between FRP(Vh) and luciferase, this study was carried out to characterize the equilibrium bindings, reductive potential, and the reversibility of the reduction of the bound FMN in the reductive half-reaction of FRP(Vh). Results indicate that, in addition to NADPH binding, NADP(+) also bound to FRP(Vh) in either the oxidized (K(d) 180 microM) or reduced (K(d) 230 microM) form. By titrations with NADP(+) and NADPH and by an isotope exchange experiment, the reduction of the bound FMN by NADPH was found to be readily reversible (K(eq) = 0.8). Hence, the reduction of FRP(Vh)-bound FMN is not the committed step in coupling the NADPH oxidation to bioluminescence. To our knowledge, such an aspect of flavin reductase catalysis has only been clearly established for FRP(Vh). Although the reductive potentials and some other properties of a R203A variant of FRP(Vh) and an NADH/NADPH-utilizing flavin reductase from Vibrio fischeri are quite similar to that of the wild-type FRP(Vh), the reversal of the reduction of bound FMN was not detected for either of these two enzymes. 相似文献
7.
Four novel amino acid type-selective triple resonance experiments to identify the backbone amino proton and nitrogen resonances of Arg and Lys and of their sequential neighbors in (13C,15N)-labeled proteins are presented: the R(i+1)-HSQC and R(i,i+1)-HSQC select signals originating from Arg side chains, the K(i+1)-HSQC and K(i,i+1)-HSQC select signals originating from Lys side chains. The selection is based on exploiting the characteristic chemical shifts of a pair of carbon atoms in Arg and Lys side chains using selective 90° pulses. The new experiments are recorded as two-dimensional 1H-15N-correlations and their performance is demonstrated with the application to a protein domain of 83 amino acids. 相似文献
8.
Zhou W Bertsova YV Feng B Tsatsos P Verkhovskaya ML Gennis RB Bogachev AV Barquera B 《Biochemistry》1999,38(49):16246-16252
The Na(+)-translocating NADH: ubiquinone oxidoreductase (Na(+)-NQR) generates an electrochemical Na(+) potential driven by aerobic respiration. Previous studies on the enzyme from Vibrio alginolyticus have shown that the Na(+)-NQR has six subunits, and it is known to contain FAD and an FeS center as redox cofactors. In the current work, the enzyme from the marine bacterium Vibrio harveyi has been purified and characterized. In addition to FAD, a second flavin, tentatively identified as FMN, was discovered to be covalently attached to the NqrC subunit. The purified V. harveyi Na(+)-NQR was reconstituted into proteoliposomes. The generation of a transmembrane electric potential by the enzyme upon NADH:Q(1) oxidoreduction was strictly dependent on Na(+), resistant to the protonophore CCCP, and sensitive to the sodium ionophore ETH-157, showing that the enzyme operates as a primary electrogenic sodium pump. Interior alkalinization of the inside-out proteoliposomes due to the operation of the Na(+)-NQR was accelerated by CCCP, inhibited by valinomycin, and completely arrested by ETH-157. Hence, the protons required for ubiquinol formation must be taken up from the outside of the liposomes, which corresponds to the bacterial cytoplasm. The Na(+)-NQR operon from this bacterium was sequenced, and the sequence shows strong homology to the previously reported Na(+)-NQR operons from V. alginolyticus and Haemophilus influenzae. Homology studies show that a number of other bacteria, including a number of pathogenic species, also have an Na(+)-NQR operon. 相似文献
9.
Yoo A Seo YS Jung JW Sung SK Kim WT Lee W Yang DR 《Journal of structural biology》2006,156(3):407-420
The 1-aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the last step in the biosynthesis of ethylene from ACC in higher plants. The complex structure of ACC oxidase/Fe(2+)/H(2)O derived from Petunia hybrida has recently been established by X-ray crystallography and it provides a vast structural information for ACC oxidase. Our mutagenesis study shows that both Lys296 and Arg299 residues in the C-terminal helix play important roles in enzyme activity. Both K296R and R299K mutant proteins retain only 30-15% of their enzyme activities with respect to that of the wild-type, implying that the positive charges of C-terminal residues are involved in enzymatic reaction. Furthermore, the sequence alignment of ACC oxidases from 24 different species indicates an existence of the exclusively conserved motif (Lys296-Glu301) especially in the C-terminus. The structure model based on our findings suggests that the positive-charged surface in the C-terminal helix of the ACC oxidase could be a major stabilizer in the spatial arrangement of reactants and that the positive-charge network between the active site and C-terminus is critical for ACC oxidase activity. 相似文献
10.
High-performance liquid chromatography mass spectrometry (HPLC MS) was employed to assess the binding behaviors of various
substrates to Vibrio harveyi chitinase A. Quantitative analysis revealed that hexaNAG preferred subsites −2 to +2 over subsites −3 to +2 and pentaNAG
only required subsites −2 to +2, while subsites −4 to +2 were not used at all by both substrates. The results suggested that
binding of the chitooligosaccharides to the enzyme essentially occurred in compulsory fashion. The symmetrical binding mode
(−2 to +2) was favored presumably to allow the natural form of sugars to be utilized effectively. Crystalline α chitin was
initially hydrolyzed into a diverse ensemble of chitin oligomers, providing a clear sign of random attacks that took place
within chitin chains. However, the progressive degradation was shown to occur in greater extent at later time to complete
hydrolysis. The effect of the reducing-end residues were also investigated by means of HPLC MS. Substitutions of Trp275 to
Gly and Trp397 to Phe significantly shifted the anomer selectivity of the enzyme toward β substrates. The Trp275 mutation
modulated the kinetic property of the enzyme by decreasing the catalytic constant (k
cat) and the substrate specificity (k
cat/K
m) toward all substrates by five- to tenfold. In contrast, the Trp397 mutation weakened the binding strength at subsite (+2),
thereby speeding up the rate of the enzymatic cleavage toward soluble substrates but slowing down the rate of the progressive
degradation toward insoluble chitin. 相似文献
11.
AIMS: To demonstrate that Vibrio harveyi produces various types of toxins and how the production of those toxins is related with luminescence. METHODS AND RESULTS: Luminescence and toxicity of eight V. harveyi were evaluated. We demonstrated that all V. harveyi emitting luminescence were isolated from marine organisms and also showed that they were highly pathogenic when compared with culture collection V. harveyi based on cytotoxic assay test. On the contrary, V. harveyi isolated from shrimp farm showed no luminescence but showed high pathogenicity based on toxicity test. The effect of protease inhibitors on pathogenicity and luminescence was also investigated. We demonstrated that light emission of pathogenic V. harveyi remarkably decreased after addition of protease inhibitor. Furthermore, extracellular proteins from cell-free culture supernatant of luminescent and nonluminescent V. harveyi were compared using SDS-PAGE analysis. Results showed that there were differences in molecular weight and amount of proteins. CONCLUSIONS: Vibrio harveyi parasiting marine organisms have both luminescence and pathogenicity. Based on this study, luminescence and protease toxin activity in V. harveyi are related. Moreover, this paper clarified that V. harveyi produces various types of toxins. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study demonstrated that V. harveyi produces two kinds of toxins, haemolysin and protease toxin. It may be clear roots of V. harveyi toxin. 相似文献
12.
The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi was purified and studied by EPR and visible spectroscopy. Two EPR signals in the NADH-reduced enzyme were detected: one, a radical signal, and the other a line around g = 1.94, which is typical for a [2Fe-2S] cluster. An E(m) of -267 mV was found for the Fe-S cluster (n = 1), independent of sodium concentration. The spin concentration of the radical in the enzyme was approximately the same under a variety of redox conditions. The time course of Na+-NQR reduction by NADH indicated the presence of at least two different flavin species. Reduction of the first species (most likely, a FAD near the NADH dehydrogenase site) was very rapid in both the presence and absence of sodium. Reduction of the second flavin species (presumably, covalently bound FMN) was slower and strongly dependent on sodium concentration, with an apparent activation constant for Na+ of approximately 3.4 mM. This is very similar to the Km for Na+ in the steady-state quinone reductase reaction catalyzed by this enzyme. These data led us to conclude that the sodium-dependent step within the Na+-NQR is located between the noncovalently bound FAD and the covalently bound FMN. 相似文献
13.
Bioluminescence in the marine bacterium Vibrio harveyi is cell density dependent and is regulated by small molecules (autoinducers) excreted by the bacteria. The autoinducer signals are relayed to a central regulator, LuxO, which acts in its phosphorylated form as a repressor of the lux operon at the early stages of cell growth. We report in these studies the purification to homogeneity of a luxO DNA binding protein (LuxT) from V. harveyi after five major chromatography steps, including a highly effective DNA affinity chromatography step and reverse-phase HPLC. Regeneration of binding activity was accomplished after HPLC and SDS-PAGE by renaturation of LuxT from guanidine hydrochloride. It was also demonstrated that the functional LuxT was a dimer of 17 kDa that bound tightly (K(d) = 2 nM) to the luxO promoter. The sequences of three tryptic peptides obtained on digestion of the purified protein did not match any sequences in the Protein Data Bank, indicating that LuxT is a new V. harveyi lux regulatory protein. 相似文献
14.
Lowery CA McKenzie KM Qi L Meijler MM Janda KD 《Bioorganic & medicinal chemistry letters》2005,15(9):2395-2398
Quorum sensing activity was investigated in the bacterium Vibrio harveyi using a series of both natural and nonnatural analogs of DPD, the penultimate precursor to autoinducer AI-2. The progression of molecules that were both synthesized and investigated includes enantiomeric variants, carbon-chain extension, and hydroxyl-functional group addition/deletions of DPD. The compilation of these studies reveals a binding cleft that can accommodate a number of different structural variants of DPD, albeit with invariably lower activities. 相似文献
15.
Atg12, a post-translational modifier, is activated and conjugated to Atg5 by a ubiquitin-like conjugation system, though it has no obvious sequence homology to ubiquitin. The Atg12-Atg5 conjugate is essential for autophagy, an intracellular bulk degradation process. Here, we show that the carboxyl-terminal region of Atg12 that is predicted to fold into a ubiquitin-like structure is necessary and sufficient for both conjugation and autophagy, which indicates that the domain essential for autophagy resides in the ubiquitin-fold region. We further show that two hydrophobic residues within the ubiquitin-fold region are important for autophagy: mutation at Y149 affects conjugate formation catalyzed by Atg10, an E2-like enzyme, while mutation at F154 has no effect on Atg12-Atg5 conjugate formation but its hydrophobic nature is essential for autophagy. In response to the F154 mutation, Atg8-PE conjugation, the other ubiquitin-like conjugation in autophagy, is severely reduced and autophagosome formation fails. Gel filtration analysis suggests that F154 plays a critical role in the assembly of a functional Atg12-Atg5.Atg16 complex that is requisite for autophagosome formation. 相似文献
16.
Bogachev AV Bertsova YV Ruuge EK Wikström M Verkhovsky MI 《Biochimica et biophysica acta》2002,1556(2-3):113-120
Two radical signals with different line widths are seen in the Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi by EPR spectroscopy. The first radical is observed in the oxidized enzyme, and is assigned as a neutral flavosemiquinone. The second radical is observed in the reduced enzyme and is assigned to be the anionic form of flavosemiquinone. The time course of Na+-NQR reduction by NADH, as monitored by stopped-flow optical spectroscopy, shows three distinct phases, the spectra of which suggest that they correspond to the reduction of three different flavin species. The first phase is fast both in the presence and absence of sodium, and is assigned to reduction of FAD to FADH2 at the NADH dehydrogenating site. The rates of the other two phases are strongly dependent on sodium concentration, and these phases are attributed to reduction of two covalently bound FMN's. Combination of the optical and EPR data suggests that a neutral FMN flavosemiquinone preexists in the oxidized enzyme, and that it is reduced to the fully reduced flavin by NADH. The other FMN moiety is initially oxidized, and is reduced to the anionic flavosemiquinone. One-electron transitions of two discrete flavin species are thus assigned as sodium-dependent steps in the catalytic cycle of Na+-NQR. 相似文献
17.
Fluorescence spectroscopic and kinetic analysis of photochemical activity, cofactor and substrate binding, and enzyme denaturation studies were performed with highly purified, recombinant pea NADPH:protochlorophyllide oxidoreductase (POR) heterologously expressed in Escherichia coli. The results obtained with an individual stereoisomer of the substrate [C8-ethyl-C13(2)-(R)-protochlorophyllide] demonstrate that the enzyme photoactive state possesses a characteristic fluorescence maximum at 646 nm that is due to the presence of specific charged amino acids in the enzyme catalytic site. The photoactive state is converted directly into an intermediate having fluorescence at 685 nm in a reaction involving direct hydrogen transfer from the cofactor (NADPH). Site-directed mutagenesis of the highly conserved Tyr275 (Y275F) and Lys279 (K279I and K279R) residues in the enzyme catalytic pocket demonstrated that the presence of these two amino acids in the wild-type POR considerably increases the probability of photoactive state formation following cofactor and substrate binding by the enzyme. At the same time, the presence of these two amino acids destabilizes POR and increases the rate of enzyme denaturation. Neither Tyr275 nor Lys279 plays a crucial role in the binding of the substrate or cofactor by the enzyme. In addition, the presence of Tyr275 is absolutely necessary for the second step of the protochlorophyllide reduction reaction, "dark" conversion of the 685 nm fluorescence intermediate and the formation of the final product, chlorophyllide. We propose that Tyr275 and Lys279 participate in the proper coordination of NADPH and PChlide in the enzyme catalytic site and thereby control the efficiency of the formation of the POR photoactive state. 相似文献
18.
Structure-function relationship in the globular type III antifreeze protein: identification of a cluster of surface residues required for binding to ice. 总被引:3,自引:1,他引:2
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H. Chao F. D. Snnichsen C. I. DeLuca B. D. Sykes P. L. Davies 《Protein science : a publication of the Protein Society》1994,3(10):1760-1769
Antifreeze proteins (AFPs) depress the freezing point of aqueous solutions by binding to and inhibiting the growth of ice. Whereas the ice-binding surface of some fish AFPs is suggested by their linear, repetitive, hydrogen bonding motifs, the 66-amino-acid-long Type III AFP has a compact, globular fold without any obvious periodicity. In the structure, 9 beta-strands are paired to form 2 triple-stranded antiparallel sheets and 1 double-stranded antiparallel sheet, with the 2 triple sheets arranged as an orthogonal beta-sandwich (Sönnichsen FD, Sykes BD, Chao H, Davies PL, 1993, Science 259:1154-1157). Based on its structure and an alignment of Type III AFP isoform sequences, a cluster of conserved, polar, surface-accessible amino acids (N14, T18, Q44, and N46) was noted on and around the triple-stranded sheet near the C-terminus. At 3 of these sites, mutations that switched amide and hydroxyl groups caused a large decrease in antifreeze activity, but amide to carboxylic acid changes produced AFPs that were fully active at pH 3 and pH 6. This is consistent with the observation that Type III AFP is optimally active from pH 2 to pH 11. At a concentration of 1 mg/mL, Q44T, N14S, and T18N had 50%, 25%, and 10% of the activity of wild-type antifreeze, respectively. The effects of the mutations were cumulative, such that the double mutant N14S/Q44T had 10% of the wild-type activity and the triple mutant N14S/T18N/Q44T had no activity. All mutants with reduced activity were shown to be correctly folded by NMR spectroscopy. Moreover, a complete characterization of the triple mutant by 2-dimensional NMR spectroscopy indicated that the individual and combined mutations did not significantly alter the structure of these proteins. These results suggest that the C-terminal beta-sheet of Type III AFP is primarily responsible for antifreeze activity, and they identify N14, T18, and Q44 as key residues for the AFP-ice interaction. 相似文献
19.
The amino terminal blocked peptide of rat liver NAD(P)H:quinone reductase (DT-diaphorase) was determined by amino acid sequence analysis and by mass spectrometry. The mature protein is composed of 273 amino acids and contains an acetylated amino terminus, which was not identified by previous cDNA analysis. The enzyme was inactivated by p-nitrobenzenesulfonyl fluoride (NBSF) or 2,4,6-trinitrobenzenesulfonate (TNBS) with pseudo-first-order kinetics. These studies suggest that essential tyrosine and lysine may be present in the active site of this enzyme. The NBSF inhibition was protected by 1-naphthol and 1-naphthylamine, but not by NAD+. However, TNBS inhibition was not prevented by the naphthalene derivatives or NAD+. Specific peptides labeled with NBSF or TNBS were isolated by high-performance liquid chromatography and were sequenced. These analyses revealed that the NBSF-labeled tyrosine resides in a predominantly hydrophobic region and TNBS-labeled lysine in a predominantly hydrophilic region. 相似文献
20.
The anticoagulant polysaccharide heparin binds and activates the plasma proteinase inhibitor antithrombin through a pentasaccharide sequence. Lys114, Lys125, and Arg129 are the three most important residues of the inhibitor for pentasaccharide binding. To elucidate to what extent another positively charged side chain can fulfill the role of each of these residues, we have mutated Lys114 and Lys125 to Arg and Arg129 to Lys. Lys114 could be reasonably well replaced with Arg with only an approximately 15-fold decrease in pentasaccharide affinity, in contrast to an approximately 10(5)-fold decrease caused by substitution with an noncharged amino acid of comparable size. However, a loss of approximately one ionic interaction on mutation to Arg indicates that the optimal configuration of the network of basic residues of antithrombin that together interact with the pentasaccharide requires a Lys in position 114. Replacement of Lys125 with Arg caused an even smaller, approximately 3-fold, decrease in pentasaccharide affinity, compared with that of approximately 400-fold caused by mutation to a neutral amino acid. An Arg in position 125 is thus essentially equivalent to the wild-type Lys in pentasaccharide binding. Substitution of Arg129 with Lys decreased the pentasaccharide affinity an appreciable approximately 100-fold, a loss approaching that of approximately 400-fold caused by substitution with a neutral amino acid. Arg is thus specifically required in position 129 for high-affinity pentasaccharide binding. This requirement is most likely due to the ability of Arg to interact with other residues of antithrombin, primarily, Glu414 and Thr44, in a manner that appropriately positions the Arg side chain for keeping the pentasaccharide anchored to the activated state of the inhibitor. 相似文献