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1.
The binding of ligands to phosphofructokinase 2 (Pfk-2) from Escherichia coli induces changes in the fluorescence emission properties of its single tryptophan residue, Trp88, suggesting that upon binding the protein undergoes a conformational change. This fluorescence probe was used to determine the presence of an allosteric site for MgATP2- in the enzyme. Fructose 6-phosphate (fructose-6-P), the first substrate that binds to the enzyme with an ordered bi-bi mechanism, increases the fluorescence up to 30%. The saturation curve for this compound is hyperbolic with a Kd of 6 microM. The titration of Pfk-2 with MgATP2- causes a quenching of fluorescence of about 30% of its initial value, with a blue shift of 7 nm in the emission maximum. The response is cooperative with a Kd of 80 microM and a Hill coefficient of 2. The interaction of MgATP2- cannot take place at the active site in the absence of fructose-6-P, due to the ordered kinetic mechanism. Addition of compounds that bind to the catalytic site of Pfk-2, such as ATP4- or Mg-AMP-PNP, did not produce significant changes in the fluorescence spectrum of Trp88. However, in the absence of Mg2+, the addition of ATP4- to the enzyme-fructose-6-P complex shows a hyperbolic increase of fluorescence of 8%. Acrylamide steady-state quenching experiments for different enzyme-ligand complexes of Pfk-2, indicate that the tryptophan in the enzyme-MgATP2- complex is exposed to a much smaller extent to the solvent than in the free enzyme or in the enzyme-fructose-6-P complex. The effect of the binding of fructose-6-P or MgATP2- on the polarization fluorescence of the emission of Trp88 in Pfk-2 indicates changes in the local mobility of the Trp88 in both enzyme complexes. The average lifetime for Trp88 in Pfk-2 was found to be unusually large, approximately 7.7 ns, and did not vary significantly with the ligation state of the enzyme, which demonstrates that the quenching or enhancement of fluorescence induced by the ligands is mainly due to the complex formation with Pfk-2. These results demonstrate the presence of an allosteric site for MgATP2- in Pfk-2 from E. coli, responsible for the inhibition of the enzyme activity by this ligand.  相似文献   

2.
BACKGROUND: Chorismate mutase (CM) catalyzes the Claisen rearrangement of chorismate to prephenate, notably the only known enzymatically catalyzed pericyclic reaction in primary metabolism. Structures of the enzyme in complex with an endo-oxabicyclic transition state analogue inhibitor, previously determined for Bacillus subtilis and Escherichia coli CM, provide structural insight into the enzyme mechanism. In contrast to these bacterial CMs, yeast CM is allosterically regulated in two ways: activation by tryptophan and inhibition by tyrosine. Yeast CM exists in two allosteric states, R (active) and t (inactive). RESULTS: We have determined crystal structures of wild-type yeast CM cocrystallized with tryptophan and an endo-oxabicyclic transition state analogue inhibitor, of wild-type yeast CM co-crystallized with tyrosine and the endo-oxabicyclic transition state analogue inhibitor and of the Thr226-->Ser mutant of yeast CM in complex with tryptophan. Binding of the transition state analogue inhibitor to CM keeps the enzyme in a 'super R' state, even if the inhibitory effector tyrosine is bound to the regulatory site. CONCLUSIONS: The endo-oxabicyclic inhibitor binds to yeast CM in a similar way as it does to the distantly related CM from E. coli. The inhibitor-binding mode supports a mechanism by which polar sidechains of the enzyme bind the substrate in the pseudo-diaxial conformation, which is required for catalytic turnover. A lysine and a protonated glutamate sidechain have a critical role in the stabilization of the transition state of the pericyclic reaction. The allosteric transition from T-->R state is accompanied by a 15 degrees rotation of one of the two subunits relative to the other (where 0 degrees rotation defines the T state). This rotation causes conformational changes at the dimer interface which are transmitted to the active site. An allosteric pathway is proposed to include residues Phe28, Asp24 and Glu23, which move toward the activesite cavity in the T state. In the presence of the transition-state analogue a super R state is formed, which is characterised by a 22 degrees rotation of one subunit relative to the other.  相似文献   

3.
The three-dimensional structures of Escherichia coli glycerol kinase (GK) with bound glycerol in the presence and absence of one of the allosteric regulators of its activity, fructose 1,6-bisphosphate (FBP), at 3.2 and 3.0 A, are presented. The molecule crystallized in space group P41212, and the structure was solved by molecular replacement. The models were refined with good stereochemistry to final R-factors of 21.1 and 21.9%, respectively. A tetrameric arrangement of monomers was observed which was essentially identical to the proposed inactive tetramer II previously described [Feese, M. D., Faber, H. R., Bystrom, C. E., Pettigrew, D. W., and Remington, S. J. (1998) Structure (in press)]. However, the crystal packing in this form was especially open, permitting the FBP binding site to be occupied and identified. The crystallographic data revealed a most unusual type of FBP binding site formed between two glycine-arginine loops (residues 234-236) where one-half of the binding site is donated by each monomer at the regulatory interface. The molecule of FBP binds in two mutually exclusive modes on a noncrystallographic 2-fold axis at 50% occupancy each; thus, a tetramer of GK binds two molecules of FBP. Ionic interactions between the 1- and 6-phosphates of FBP and Arg 236 were observed in addition to hydrogen bonding interactions between the backbone amide of Gly 234 and the 6-phosphate. No contacts between the protein and the furanose ring were observed. Mutagenesis of Arg 236 to alanine drastically reduced the extent of inhibition of GK by FBP and lowered, but did not eliminate, the ability of FBP to promote tetramer association. These observations are consistent with the previously characterized mechanism of FBP inhibition of GK, in which FBP acts both to promote dimer-tetramer assembly and to inactivate the tetramers.  相似文献   

4.
The histidine phosphocarrier protein (HPr) is an essential element in sugar transport by the bacterial phosphoenolpyruvate:sugar phosphotransferase system. Ligand fishing, using surface plasmon resonance, was used to show the binding of HPr to a nonphosphotransferase protein in extracts of Escherichia coli; the protein was subsequently identified as glycogen phosphorylase (GP). The high affinity (association constant approximately 10(8) M-1), species-specific interaction was also demonstrated in electrophoretic mobility shift experiments by polyacrylamide gel electrophoresis. Equilibrium ultracentrifugation analysis indicates that HPr allosterically regulates the oligomeric state of glycogen phosphorylase. HPr binding increases GP activity to 250% of the level in control assays. Kinetic analysis of coupled enzyme assays shows that the binding of HPr to GP causes a decrease in the Km for glycogen and an increase in the Vmax for phosphate, indicating a mixed type activation. The stimulatory effect of E. coli HPr on E. coli GP activity is species-specific, and the unphosphorylated form of HPr activates GP more than does the phosphorylated form. Replacement of specific amino acids in HPr results in reduced GP activation; HPr residues Arg-17, Lys-24, Lys-27, Lys-40, Ser-46, Gln-51, and Lys-72 were established to be important. This novel mechanism for the regulation of GP provides the first evidence directly linking E. coli HPr to the regulation of carbohydrate metabolism.  相似文献   

5.
BACKGROUND: Microbial pectin and pectate lyases are virulence factors that degrade the pectic components of the plant cell wall. The homogalacturan backbone of pectin varies in its degree of methylation from the highly methylated and relatively hydrophobic form known as pectin, to the fully demethylated and highly charged form known as pectate. Methylated and demethylated regions of pectin are cleaved by pectin lyase and calcium-dependent pectate lyases, respectively. Protein engineering of lyases specific for particular patterns of methylation, will yield modified pectins of high value to the food and pharmaceutical industries. RESULTS: The crystal structures of pectin lyase A from two strains of Aspergillus niger, N400 and 4M-147, have been determined at pH 6.5 (2.4 A resolution) and pH 8.5 (1.93 A resolution), respectively. The structures were determined by a combination of molecular replacement, multiple isomorphous replacement and intercrystal averaging. Pectin lyase A folds into a parallel beta helix and shares many of the structural features of pectate lyases, despite no more than 17% sequence identity after pairwise structure-based alignment. These shared structural features include amino acid stacks and the asparagine ladder. However, the differences in the substrate-binding clefts of these two enzymes are striking. In pectin lyase A, the cleft is dominated by aromatic residues and is enveloped by negative electrostatic potential. In pectate lyases, this cleft is rich in charged residues and contains an elongated ribbon of positive potential when Ca2+ is bound. The major difference between the two pectin lyase A structures from the two strains is in the conformation of the loop formed by residues 182-187. These observed differences are due to the different pH values of crystallization. CONCLUSIONS: The substrate-binding clefts and catalytic machinery of pectin and pectate lyases have diverged significantly. Specificity is dictated by both the nature of the protein-carbohydrate interaction and long-range electrostatic forces. Three potential catalytic residues have been identified in pectin lyase, two of these are common to pectate lyases. Pectin lyase A does not bind Ca2+ but an arginine residue is found in an equivalent position to the Ca2+ ion in pectate lyase, suggesting a similar role in catalysis. The activity of pectin lyase A is pH -dependent with an optimum activity at pH 5.5. The activity drops above pH 7.0 due to a conformational change at the binding cleft, triggered by the proximity of two buried aspartate residues.  相似文献   

6.
Molecular chaperones are essential to all living organisms. Their key role consists of mediating protein folding within the cell. Recent functional studies have provided more detailed information about the function and regulation of the chaperone network. Highlights of the past year include the crystal structure determinations of the asymmetric GroEL-GroES complex and of their isolated peptide-binding domains.  相似文献   

7.
The Escherichia coli purine repressor, PurR, exists in an equilibrium between open and closed conformations. Binding of a corepressor, hypoxanthine or guanine, shifts the allosteric equilibrium in favor of the closed conformation and increases the operator DNA binding affinity by 40-fold compared to aporepressor. Glu70 and Trp147 PurR mutations were isolated which perturb the allosteric equilibrium. Three lines of evidence indicate that the allosteric equilibrium of E70A and W147A aporepressors was shifted toward the closed conformation. First, compared to wild-type PurR, these mutant repressors had a 10-30-fold higher corepressor binding affinity. Second, the mutant aporepressors bound to operator DNA with an affinity that is characteristic of the wild-type PurR holorepressor. Third, binding of guanine to wild-type PurR resulted in a near-UV circular dichroism spectral change at 297-305 nm that is attributed to the closed conformation. The circular dichroism spectrum of the E70A aporepressor at 297-305 nm was that expected for the closed conformation, and it was not appreciably altered by corepressor binding. Mutational analysis was used to identify an Arg115-Ser46' interdomain intersubunit hydrogen bond that is necessary for transmitting the allosteric transition in the corepressor binding domain to the DNA binding domain. R115A and S46G PurR mutants were defective in DNA binding in vitro and repressor function in vivo although corepressor binding was identical to the wild type. These results establish that the hydrogen bond between the side chain NH2 of Arg115 and the main chain CO of Ser46' plays a critical role in interdomain signaling.  相似文献   

8.
Exposure to continuous darkness and chronic treatment with melatonin, for six weeks, stimulated reproduction in the male Indian desert gerbil as evidenced by morphometric data. Exposure to continuous light, for same duration, on the other hand inhibited reproduction. The results are opposite to those reported from similar studies on temperate zone species. Surface areas of abdominal scent glands increased following both, exposures to continuous darkness and the treatment with melatonin. Exposure to continuous light decreased the scent gland surface area. Assessment of scent gland activity could be useful in evaluation of reproductive function as they are dependent on sex steroids. Melatonin that mediates photoperiodic influence on reproduction is not always inhibitory to gonads.  相似文献   

9.
CMP kinases from Bacillus subtilis and from Escherichia coli are encoded by the cmk gene (formerly known as jofC in B. subtilis and as mssA in E. coli). Similar in their primary structure (43% identity and 67% similarity in amino acid sequence), the two proteins exhibit significant differences in nucleotide binding and catalysis. ATP, dATP, and GTP are equally effective as phosphate donors with E. coli CMP kinase whereas GTP is a poor substrate with B. subtilis CMP kinase. While CMP and dCMP are the best phosphate acceptors of both CMP kinases, the specific activity with these substrates and ATP as donor are 7- to 10-fold higher in the E. coli enzyme; the relative Vm values with UMP and CMP are 0.1 for the B. subtilis CMP kinase and 0.01 for the E. coli enzyme. CMP increased the affinity of E. coli CMP kinase for ATP or for the fluorescent analog 3'-anthraniloyl dATP by one order of magnitude but had no effect on the B. subtilis enzyme. The differences in the catalytic properties of B. subtilis and E. coli CMP kinases might be reflected in the structure of the two proteins as inferred from infrared spectroscopy. Whereas the spectrum of B. subtilis CMP kinase is dominated by a band at 1633 cm-1 (representing beta type structures), the spectrum of the E. coli enzyme is dominated by two bands at 1653 and 1642 cm-1 associated with alpha-helical and unordered structures, respectively. CMP induced similar spectral changes in both proteins with a rearrangement of some of the beta-structures. ATP increases the denaturation temperature of B. subtilis CMP kinase by 9.3 degrees C, whereas in the case of the E. coli enzyme, binding of ATP has only a minor effect.  相似文献   

10.
Six single-Trp mutants were engineered by individually reintroducing each of the native Trp residues into a functional lactose permease mutant devoid of Trp (Trp-less permease; Menezes ME, Roepe PD, Kaback HR, 1990, Proc Natl Acad Sci USA 87:1638-1642), and fluorescent properties were studied with respect to solvent accessibility, as well as alterations produced by ligand binding. The emission of Trp 33, Trp 78, Trp 171, and Trp 233 is strongly quenched by both acrylamide and iodide, whereas Trp 151 and Trp 10 display a decrease in fluorescence in the presence of acrylamide only and no quenching by iodide. Of the six single-Trp mutants, only Trp 33 exhibits a significant change in fluorescence (ca. 30% enhancement) in the presence of the substrate analog beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). This effect was further characterized by site-directed fluorescent studies with purified single-Cys W33-->C permease labeled with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS). Titration of the change in the fluorescence spectrum reveals a 30% enhancement accompanied with a 5-nm blue shift in the emission maximum, and single exponential behavior with an apparent KD of 71 microM. The effect of substrate binding on the rate of MIANS labeling of single-Cys 33 permease was measured in addition to iodide and acrylamide quenching of the MIANS-labeled protein. Complete blockade of labeling is observed in the presence of TDG, as well as a 30% decrease in accessibility to iodide with no change in acrylamide quenching. Overall, the findings are consistent with the proposal (Wu J, Frillingos S, Kaback HR, 1995a, Biochemistry 34:8257-8263) that ligand binding induces a conformational change at the C-terminus of helix I such that Pro 28 and Pro 31, which are on one face, become more accessible to solvent, whereas Trp 33, which is on the opposite face, becomes less accessible to the aqueous phase. The findings regarding accessibility to collisional quenchers are also consistent with the predicted topology of the six native Trp residues in the permease.  相似文献   

11.
The wild-type TMP kinases from Escherichia coli and from a strain hypersensitive to 5-bromo-2'-deoxyuridine were characterized comparatively. The mutation at codon 146 causes the substitution of an alanine residue for glycine in the enzyme, which is accompanied by changes in the relative affinities for 5-Br-UMP and TMP compared to those of the wild-type TMP kinase. Plasmids carrying the wild-type tmk gene from Escherichia coli or Bacillus subtilis, but not the defective tmk gene, restored the resistance to bromodeoxyuridine of an E. coli mutant strain.  相似文献   

12.
The fundamental conformational states of right-handed double helical DNA, the A- and B-forms, are associated with distinct puckers of the sugar moieties. The furanose conformation itself is affected by the steric and electronic nature of the ring substituents. For example, a strongly electronegative substituent at the C2' position, such as in the 2'-deoxy-2'-fluoro ribo furanosyl analogue, will drive the conformational equilibrium towards the C3'- endo type (north). Conversely, the 2'-deoxy-2'-fluoro arabino furanosyl modification with opposite stereochemistry at C2' appears to have a preference for a C2'- endo type pucker (south). Incorporation of 2'-fluoroarabinofuranosyl thymines was previously shown to enhance the thermodynamic stability of B-DNA duplexes. We have determined the crystal structures of the B-DNA dodecamer duplexes [d(CGCGAASSCGCG)]2and [d(CGCGAASTCGCG)]2with incorporated 2'-deoxy-2'-fluoroarabinofuranosyl thymines S (south) at 1.55 A resolution. In the crystal structures, all S residues adopt an O4'- endo conformation (east), well compatible with an overall B-form duplex geometry. In addition to the increased rigidity of S nucleosides, a clathrate-like ordered water structure around the 2'-fluorines may account for the observed larger thermodynamic stability of DNA duplexes containing 2'-deoxy-2'-fluoroarabino thymidines.  相似文献   

13.
The three-dimensional structures of four mutant E. coli inorganic pyrophosphatases (PPases) with single Asp-->Asn substitutions at positions 42, 65, 70, and 97 were solved at 1.95, 2.15, 2.10, and 2.20 A resolution, respectively. Asp-42-->Asn and Asp-65-->Asn mutant PPases were prepared as complexes with sulfate--a structural analog of phosphate, the product of enzymatic reaction. A comparison of mutant enzymes with native PPases revealed that a single amino acid substitution changes the position of the mutated residue as well as the positions of several functional groups and some parts of a polypeptide chain. These changes are responsible for the fact that mutant PPases differ from the native ones in their catalytic properties. The sulfate binding to the mutant PPase active site causes molecular asymmetry, as shown for the native PPase earlier. The subunit asymmetry is manifested in different positions of sulfate and several functional groups, as well as changes in packing of hexamers in crystals and in cell parameters.  相似文献   

14.
The heat shock response of Escherichia coli is regulated by the cellular level and the activity of sigma32, an alternative sigma factor for heat shock promoters. FtsH, a membrane-bound AAA-type metalloprotease, degrades sigma32 and has a central role in the control of the sigma32 level. The ftsH null mutant was isolated, and establishment of the DeltaftsH mutant allowed us to investigate control mechanisms of the stability and the activity of sigma32 separately in vivo. Loss of the FtsH function caused marked stabilization and consequent accumulation of sigma32 ( approximately 20-fold of the wild type), leading to the impaired downregulation of the level of sigma32. Surprisingly, however, DeltaftsH cells express heat shock proteins only two- to threefold higher than wild-type cells, and they also show almost normal heat shock response upon temperature upshift. These results indicate the presence of a control mechanism that downregulates the activity of sigma32 when it is accumulated. Overproduction of DnaK/J reduces the activity of sigma32 in DeltaftsH cells without any detectable changes in the level of sigma32, indicating that the DnaK chaperone system is responsible for the activity control of sigma32 in vivo. In addition, CbpA, an analogue of DnaJ, was demonstrated to have overlapping functions with DnaJ in both the activity and the stability control of sigma32.  相似文献   

15.
Changes in the kinetic properties of voltage-activated sodium currents (I(Na)) were studied in rat retinal ganglion cells during in vivo differentiation. Whole-cell recordings from cells maintained as retinal slices or whole-mounts were examined using the patch-clamp technique in the perforated patch mode. Voltage-clamp recordings revealed significant ontogenetic modifications in key properties of I(Na) and the present study described for the first time the detailed time course of such alterations. I(Na) was first expressed on embryonic day 17/18 (E17/18). Current density increased during development from an average of -81 pA/pF on E17/18 to a maximum of -747pA/pF on postnatal day 10/12 (P10/12). Simultaneously, the activation of I(Na) shifted towards more negative potentials, reflected by a shift in the potential of half-activation from -14.1 mV on E17/18 to - 37.5 mV on P10/12. No significant changes in these parameters were observed after P10/12. Steady-state inactivation shifted first towards more positive potentials, reflected by a shift in the potential of half-inactivation from -51 mV on E17/18 to -38 mV on P3/5, but shifted back towards more negative values thereafter (-44 mV in the adult). The most striking feature of I(Na) in rat RGCs was a transient slowing of I(Na) kinetics that was never described before. Time to peak and decay time constants increased between E20 and P5, resulting in slow and broad sodium currents within a developmental period that is characterized by intensive synaptogenesis in the target structures of retinal ganglion cells and maximum retinal ganglion cell death. Thereafter, time to peak and decay time constants decreased again to values found before E20, resulting in rapid sodium spikes. In conclusion, sodium currents in rat retinal ganglion cells displayed substantial electrophysiological changes during pre- and postnatal development. These changes in the sodium system had different temporal time patterns, indicating that they may play specific roles during the development of the visual system.  相似文献   

16.
PURPOSE: We have previously reported incomplete cytotoxic responses of other human solid tumors (bladder, head and neck, ovarian and prostate) to paclitaxel. This finding is qualitatively different from the nearly complete response observed in monolayer cultures of human cancer cell lines. The present study examined the pharmacodynamics of paclitaxel in human breast tumors. METHODS: Three-dimensional histocultures of patient tumors were used. The cytostatic effect was evaluated by measurement of the inhibition of 48-h cumulative bromodeoxyuridine (BrdUrd) incorporation. The apoptotic effect was evaluated in terms of morphological changes and by in situ DNA end labeling. RESULTS: Paclitaxel produced partial cytostasis (approximately 30% maximum) and induced apoptosis (maximum apoptotic index of 3.3% to 29%) in all 15 tumors. More than 95% of apoptotic cells were BrdUrd labeled, but not all BrdUrd-labeled cells were apoptotic. The maximal apoptotic indices in the tumors were significantly correlated with the BrdUrd labeling index of untreated controls (r2 = 0.63, P < 0.01). The maximum apoptotic effect was observed at a tenfold lower drug concentration (0.1 microM) compared to the maximum cytostatic effect (1 microM). Neither of these effects was enhanced by increasing the drug concentration to 10 microM. CONCLUSIONS: The pharmacodynamics of paclitaxel in human breast tumors are comparable to those found in other human solid tumors. The labeling of apoptotic cells by BrdUrd and the correlation between the proliferation index and apoptosis suggest that drug-induced apoptosis is linked to cell proliferation and is completed after DNA synthesis. The finding that maximal cytostatic and apoptotic effects of paclitaxel were achieved at or below the clinically achievable concentration of 1 microM suggests further increasing the dose to elevate plasma concentration beyond 1 microM may not improve treatment outcome.  相似文献   

17.
18.
The carbon monoxide compounds of the fully reduced and mixed valence forms of cytochrome bo from Escherichia coli were laser photolysed under anaerobic conditions at room temperature. The carbon monoxide recombined with characteristic rate constants of 50 s-1 or 35 s-1 in the fully reduced and mixed valence forms, respectively. Rates of CO recombination with the fully reduced enzyme were examined in a variety of mutant forms of cytochrome bo, produced by site-directed mutagenesis. A method was developed to deconvolute cytochromes bo and bd, leading to some reassessment of histidine ligands to the metals. Significant changes in the rate constant of recombination of carbon monoxide occurred in many of these mutants and these results could be rationalised generally in terms of our current working model of the folding structure of subunit I. In the mixed valence form of the enzyme the transient photolysis spectra in the visible region are consistent with a rapid electron redistribution from the binuclear centre to the low-spin haem. This electron transfer is biphasic, with rate constants of around 10(5) and 8000 s-1. The process was also examined in the His-333-Leu mutant, in which a putative histidine ligand to CuB is replaced by leucine, and which results in the loss of the CuB. It appeared that rapid haem-haem electron transfer could still occur. The observation that CuB is apparently not required for rapid haem-haem electron transfer is consistent with the recently proposed model in which the two haems are positioned on opposite sides of transmembrane helix X in subunit I of the oxidase.  相似文献   

19.
The bivalve mollusc Lucina pectinata harbors sulfide-oxidizing chemoautotrophic bacteria and expresses a monomeric hemoglobin I, HbI, with normal O2, but extraordinarily high sulfide affinity. The crystal structure of aquomet Lucina HbI has revealed an active site with three residues not commonly found in vertebrate globins: Phe(B10), Gln(E7), and Phe(E11) (Rizzi, M., Wittenberg, J. B., Coda, A., Fasano, M., Ascenzi, P., and Bolognesi, M. (1994) J. Mol. Biol. 244, 86-89). Engineering these three residues into sperm whale myoglobin results in a triple mutant with approximately 700-fold higher sulfide affinity than for wild-type. The single crystal x-ray structure of the aquomet derivative of the myoglobin triple mutant and the solution 1H NMR active site structures of the cyanomet derivatives of both the myoglobin mutant and Lucina HbI have been determined to examine further the structural origin of their unusually high sulfide affinities. The major differences in the distal pocket is that in the aquomet form the carbonyl of Gln64(E7) serves as a H-bond acceptor, whereas in the cyanomet form the amido group acts as H-bond donor to the bound ligand. Phe68(E11) is rotated approximately 90 degrees about chi2 and located approximately 1-2 A closer to the iron atom in the myoglobin triple mutant relative to its conformation in Lucina HbI. The change in orientation potentially eliminates the stabilizing interaction with sulfide and, together with the decrease in size of the distal pocket, accounts for the 7-fold lower sulfide affinity of the myoglobin mutant compared with that of Lucina HbI.  相似文献   

20.
The structures of AmpC beta-lactamase from Escherichia coli, alone and in complex with a transition-state analogue, have been determined by X-ray crystallography. The native enzyme was determined to 2.0 A resolution, and the structure with the transition-state analogue m-aminophenylboronic acid was determined to 2.3 A resolution. The structure of AmpC from E. coli resembles those previously determined for the class C enzymes from Enterobacter cloacae and Citrobacter freundii. The transition-state analogue, m-aminophenylboronic acid, makes several interactions with AmpC that were unexpected. Perhaps most surprisingly, the putative "oxyanion" of the boronic acid forms what appears to be a hydrogen bond with the backbone carbonyl oxygen of Ala318, suggesting that this atom is protonated. Although this interaction has not previously been discussed, a carbonyl oxygen contact with the putative oxyanion or ligand carbonyl oxygen appears in most complexes involving a beta-lactam recognizing enzyme. These observations may suggest that the high-energy intermediate for amide hydrolysis by beta-lactamases and related enzymes involves a hydroxyl and not an oxyanion, although the oxyanion form certainly cannot be discounted. The involvement of the main-chain carbonyl in ligand and transition-state recognition is a distinguishing feature between serine beta-lactamases and serine proteases, to which they are often compared. AmpC may use the interaction between the carbonyl of Ala318 and the carbonyl of the acylated enzyme to destabilize the ground-state intermediate, this destabilization energy might be relieved in the transition state by a hydroxyl hydrogen bond. The structure of the m-aminophenylboronic acid adduct also suggests several ways to improve the affinity of this class of inhibitor and points to the existence of several unusual binding-site-like features in the region of the AmpC catalytic site.  相似文献   

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