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1.
The X-ray crystal structure of human soluble epoxide hydrolase (sEH) has been determined at 2.6 A resolution, revealing a domain-swapped quaternary structure identical to that observed for the murine enzyme [Argiriadi, M. A., Morisseau, C., Hammock, B. D., and Christianson, D. W. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10637-10642]. As with the murine enzyme, the epoxide hydrolytic mechanism of the human enzyme proceeds through an alkyl-enzyme intermediate with Asp-333 in the C-terminal domain. The structure of the human sEH complex with N-cyclohexyl-N'-(iodophenyl)urea (CIU) has been determined at 2.35 A resolution. Tyr-381 and Tyr-465 donate hydrogen bonds to the alkylurea carbonyl group of CIU, consistent with the proposed roles of these residues as proton donors in the first step of catalysis. The N-terminal domain of mammalian sEH contains a 15 A deep cleft, but its biological function is unclear. Recent experiments demonstrate that the N-terminal domain of human sEH catalyzes the metal-dependent hydrolysis of phosphate esters [Cronin, A., Mowbray, S., Dürk, H., Homburg, S., Fleming, I., Fisslthaler, B., Oesch, F., and Arand, M. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 1552-1557; Newman, J. W., Morisseau, C., Harris, T. R., and Hammock, B. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 1558-1563]. The binding of Mg(2+)-HPO4(2-) to the N-terminal domain of human sEH in its CIU complex reveals structural features relevant to those of the enzyme-substrate complex in the phosphatase reaction. 相似文献
2.
Mechanistic and metabolic inferences from the binding of substrate analogues and products to arginase 总被引:2,自引:0,他引:2
Cox JD Cama E Colleluori DM Pethe S Boucher JL Mansuy D Ash DE Christianson DW 《Biochemistry》2001,40(9):2689-2701
Arginase is a binuclear Mn(2+) metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. X-ray crystal structures of arginase complexed to substrate analogues N(omega)-hydroxy-L-arginine and N(omega)-hydroxy-nor-L-arginine, as well as the products L-ornithine and urea, complete a set of structural "snapshots" along the reaction coordinate of arginase catalysis when interpreted along with the X-ray crystal structure of the arginase-transition-state analogue complex described in Kim et al. [Kim, N. N., Cox, J. D., Baggio, R. F., Emig, F. A., Mistry, S., Harper, S. L., Speicher, D. W., Morris, Jr., S. M., Ash, D. E., Traish, A. M., and Christianson, D. W. (2001) Biochemistry 40, 2678-2688]. Taken together, these structures render important insight on the structural determinants of tight binding inhibitors. Furthermore, we demonstrate for the first time the structural mechanistic link between arginase and NO synthase through their respective complexes with N(omega)-hydroxy-L-arginine. That N(omega)-hydroxy-L-arginine is a catalytic intermediate for NO synthase and an inhibitor of arginase reflects the reciprocal metabolic relationship between these two critical enzymes of L-arginine catabolism. 相似文献
3.
D J Neidhart P L Howell G A Petsko V M Powers R S Li G L Kenyon J A Gerlt 《Biochemistry》1991,30(38):9264-9273
The crystal structure of mandelate racemase (MR) has been solved at 3.0-A resolution by multiple isomorphous replacement and subsequently refined against X-ray diffraction data to 2.5-A resolution by use of both molecular dynamics refinement (XPLOR) and restrained least-squares refinement (PROLSQ). The current crystallographic R-factor for this structure is 18.3%. MR is composed of two major structural domains and a third, smaller, C-terminal domain. The N-terminal domain has an alpha + beta topology consisting of a three-stranded antiparallel beta-sheet followed by an antiparallel four alpha-helix bundle. The central domain is a singly wound parallel alpha/beta-barrel composed of eight central strands of beta-sheet and seven alpha-helices. The C-terminal domain consists of an irregular L-shaped loop with several short sections of antiparallel beta-sheet and two short alpha-helices. This C-terminal domain partially covers the junction between the major domains and occupies a region of the central domain that is filled by an eight alpha-helix in all other known parallel alpha/beta-barrels except for the barrel domain in muconate lactonizing enzyme (MLE) [Goldman, A., Ollis, D. L., & Steitz, T. A. (1987) J. Mol. Biol. 194, 143] whose overall polypeptide fold and amino acid sequence are strikingly similar to those of MR [Neidhart, D. J., Kenyon, G. L., Gerlt, J. A., & Petsko, G. A. (1990) Nature 347, 692]. In addition, the crystal structure reveals that, like MLE, MR is tightly packed as an octamer of identical subunits. The active site of MR is located between the two major domains, at the C-terminal ends of the beta-strands in the alpha/beta-barrel domain. The catalytically essential divalent metal ion is ligated by three side-chain carboxyl groups contributed by residues of the central beta-sheet. A model of a productive substrate complex of MR has been constructed on the basis of difference Fourier analysis at 3.5-A resolution of a complex between MR and (R,S)-p-iodomandelate, permitting identification of residues that may participate in substrate binding and catalysis. The ionizable groups of both Lys 166 and His 297 are positioned to interact with the chiral center of substrate, suggesting that both of these residues may function as acid/base catalysts.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
4.
Cama E Colleluori DM Emig FA Shin H Kim SW Kim NN Traish AM Ash DE Christianson DW 《Biochemistry》2003,42(28):8445-8451
Arginase is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to form l-ornithine and urea. The X-ray crystal structure of a fully active, truncated form of human arginase II complexed with a boronic acid transition state analogue inhibitor has been determined at 2.7 A resolution. This structure is consistent with the hydrolysis of l-arginine through a metal-activated hydroxide mechanism. Given that human arginase II appears to play a role in regulating l-arginine bioavailability to NO synthase in human penile corpus cavernosum smooth muscle, the inhibition of human arginase II is a potential new strategy for the treatment of erectile dysfunction [Kim, N. N., Cox, J. D., Baggio, R. F., Emig, F. A., Mistry, S., Harper, S. L., Speicher, D. W., Morris, S. M., Ash, D. E., Traish, A. M., and Christianson, D. W. (2001) Biochemistry 40, 2678-2688]. Since NO synthase is found in human clitoral corpus cavernosum and vagina, we hypothesized that human arginase II is similarly present in these tissues and functions to regulate l-arginine bioavailability to NO synthase. Accordingly, hemodynamic studies conducted with a boronic acid arginase inhibitor in vivo are summarized, suggesting that the extrahepatic arginase plays a role in both male and female sexual arousal. Therefore, arginase II is a potential target for the treatment of male and female sexual arousal disorders. 相似文献
5.
An interactive computer graphics study of thermolysin-catalyzed peptide cleavage and inhibition by N-carboxymethyl dipeptides 总被引:12,自引:0,他引:12
Interactive computer graphics was used as a tool in studying the cleavage mechanism of the model substrate Z-Phe-Phe-Leu-Trp by the zinc endopeptidase thermolysin. Two Michaelis complexes and three binding orientations of the tetrahedral intermediate to the crystal structure of thermolysin were investigated. Our results indicate that a Michaelis complex, which does not involve coordination of the scissile peptide to the zinc, is consistent with available experimental data and the most plausible of the two complexes. A tetrahedral intermediate complex wherein the two oxygens of the hydrated scissile peptide straddle the zinc in a bidentate fashion results in the most favorable interactions with the active site. The preferred tetrahedral intermediate and Michaelis complex provide a rationalization for the published substrate data. A trajectory for proceeding from the Michaelis complex to the tetrahedral intermediate is proposed. This trajectory involves a simultaneous activation of the zinc-bound water molecule concurrent with attack on the scissile peptide. A detailed ordered product release mechanism is also presented. These studies suggest some modifications and a number of extensions to the mechanism proposed earlier [Kester, W. R., & Matthews, B. W. (1977) Biochemistry 16, 2506; Holmes, M. A., & Matthews, B. W. (1981) Biochemistry 20, 6912]. The binding mode of the thermolysin inhibitor N-(1-carboxy-3-phenylpropyl)-L-leucyl-L-tryptophan [Monzingo, A. F., & Matthews, B. W. (1984) Biochemistry (preceding paper in this issue)] is compared with that of the preferred tetrahedral intermediate, providing insight into this inhibitor design. 相似文献
6.
《Ibis》1989,131(2):307-312
C ollar , N.J. & A ndrew , P. 1988. Birds to Watch: The ICBP Check-list of Threatened Birds.
D allmann , M. 1987. Der Zaunkönig.
F iuczynski , D. 1987. Der Baumfalke. Die Neue Brehm-Bücherei No. 575.
F ry , C.H., K eith , S. & U rban , E.K. 1988. The Birds of Africa, Vol. III.
F urness , R.W. 1987. The Skuas.
H ölzinger , J. 1987. Die Vögel Baden-Württembergs 1 (Parts 1–3).
J ohnson , T.H. 1988. Biodiversity and Conservation in the Caribbean: Profiles of Selected Islands.
K laus , S., A ndreev , A.V., B ergmann , H.-H., M üller , F., P orkert , J. & W iesner , J. 1986. Die Auerhiihner. Die Neue Brehm-Biicherei No. 86.
P yle , P., H owell , S.N.G., Y unick , R.P. & D e S ante , D.F. 1987. Identification Guide to North American Passerines.
S now , D. & B. 1988. Birds and Berries.
SOVON 1987 (eds. J. B ekhuis (et al.). Atlas van de Nederlandse Vogels.
Nederlandse Broedvogels (1979). A long English summary at the end enables readers unfamiliar with Dutch to extract the maximum possible amount of information from this impressive work.
T oivanen , A. & T oivanen , P. (eds) Avian Immunology: Basis and Practice. 2 volumes. 相似文献
D allmann , M. 1987. Der Zaunkönig.
F iuczynski , D. 1987. Der Baumfalke. Die Neue Brehm-Bücherei No. 575.
F ry , C.H., K eith , S. & U rban , E.K. 1988. The Birds of Africa, Vol. III.
F urness , R.W. 1987. The Skuas.
H ölzinger , J. 1987. Die Vögel Baden-Württembergs 1 (Parts 1–3).
J ohnson , T.H. 1988. Biodiversity and Conservation in the Caribbean: Profiles of Selected Islands.
K laus , S., A ndreev , A.V., B ergmann , H.-H., M üller , F., P orkert , J. & W iesner , J. 1986. Die Auerhiihner. Die Neue Brehm-Biicherei No. 86.
P yle , P., H owell , S.N.G., Y unick , R.P. & D e S ante , D.F. 1987. Identification Guide to North American Passerines.
S now , D. & B. 1988. Birds and Berries.
SOVON 1987 (eds. J. B ekhuis (et al.). Atlas van de Nederlandse Vogels.
Nederlandse Broedvogels (1979). A long English summary at the end enables readers unfamiliar with Dutch to extract the maximum possible amount of information from this impressive work.
T oivanen , A. & T oivanen , P. (eds) Avian Immunology: Basis and Practice. 2 volumes. 相似文献
7.
Yu SS Ji CZ Wu YP Lee TL Lai CH Lin SC Yang ZL Wang VC Chen KH Chan SI 《Biochemistry》2007,46(48):13762-13774
The crystal structure of the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) has been reported recently [Lieberman, R. L., and Rosenzweig, A. C. (2005) Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane, Nature 434, 177-182]. Subsequent work has shown that the preparation on which the X-ray analysis is based might be missing many of the important metal cofactors, including the putative trinuclear copper cluster at the active site as well as ca. 10 copper ions (E-clusters) that have been proposed to serve as a buffer of reducing equivalents to re-reduce the copper atoms at the active site following the catalytic chemistry [Chan, S. I., Wang, V. C.-C., Lai, J. C.-H., Yu, S. S.-F., Chen, P. P.-Y., Chen, K. H.-C., Chen, C.-L., and Chan, M. K. (2007) Redox potentiometry studies of particulate methane monooxygenase: Support for a trinuclear copper cluster active site, Angew. Chem., Int. Ed. 46, 1992-1994]. Since the aqueous-exposed domains of the 45 kDa subunit (PmoB) have been suggested to be the putative binding domains for the E-cluster copper ions, we have cloned and overexpressed in Escherichia coli the two aqueous-exposed subdomains toward the N- and C-termini of the subunit: the N-terminal subdomain (residues 54-178) and the C-terminal subdomain (residues 257-394 and 282-414). The recombinant C-terminal water-exposed subdomain is shown to behave like a Cu(I) sponge, taking up to ca. 10 Cu(I) ions cooperatively when cupric ions are added to the protein fragment in the presence of dithiothreitol or ascorbate. In addition, circular dichroism measurements reveal that the C-terminal subdomain folds into a beta-sheet structure in the presence of Cu(I). The propensity for the C-terminal subdomain to bind Cu(I) is consistent with the high redox potential(s) determined for the E-cluster copper ions in the pMMO. These properties of the E-clusters are in accordance with the function proposed for these copper ions in the turnover cycle of the enzyme. 相似文献
8.
Human cathepsin D (hCatD) is an aspartic peptidase with a low pH optimum. X-ray crystal structures have been solved for an active, low pH (pH 5.1) form (CatD(lo)) [Baldwin, E. T., Bhat, T. N., Gulnik, S., Hosur, M. V., Sowder, R. C., Cachau, R. E., Collins, J., Silva, A. M., and Erickson, J. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6796-6800] and an inactive, high pH (pH 7.5) form (CatD(hi)) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. It has been suggested that ionizable switches involving the carboxylate side chains of E5, E180, and D187 may mediate the reversible interconversion between CatD(hi) and CatD(lo) and that Y10 stabilizes CatD(hi) [Lee, A. Y., Gulnik, S. V., and Erickson, J. W. (1998) Nat. Struct. Biol. 5, 866-871]. To test these hypotheses, we generated single point mutants in "short" recombinant human pseudocathepsin D (srCatD), a model kinetically similar to hCatD [Beyer, B. M., and Dunn, B. M. (1996) J. Biol. Chem. 271, 15590-15596]. E180Q, Y10F, and D187N exhibit significantly higher kcat/Km values (2-, 3-, and 6-fold, respectively) at pH 3.7 and 4.75 compared to srCatD, indicating that these residues are important in stabilizing the CatD(hi). E5Q exhibits a 2-fold lower kcat/Km compared to srCatD at both pH values, indicating the importance of E5 in stabilizing the CatD(lo). Accordingly, full time-course "pH-jump" (pH 5.5-4.75) studies of substrate hydrolysis indicate that E180Q, D187N, and Y10F have shorter kinetic lag phases that represent the change from CatD(hi) to CatD(lo) compared to srCatD and E5Q. Intrinsic tryptophan fluorescence reveals that the variants have a native-like structure over the pH range of our assays. The results indicate that E180 and D187 participate as an electrostatic switch that initiates the conformational change of CatD(lo) to CatD(hi) and Y10 stabilizes CatD(hi) by hydrogen bonding to the catalytic Asp 33. E5 appears to play a less significant role as an ionic switch that stabilizes CatD(lo). 相似文献
9.
The complete sequence of histone 2B of Drosophila has been determined by using an improved Beckman sequenator. Comparing these data with those previously published by other investigators on the histone 2B of calf [Iwai, K., Hayashi, H., & Ishikawa, K. (1972) J. Biochem. (Tokyo) 72, 357--367], trout [Koostra, A., & Bailey, G. S. (1978) Biochemistry 17, 2504--2510], and Patella (a limpet) [van Helden, P. D., Strickland, W. N., Brandt, W. F., & von Holt, C. (1979) Eur. J. Biochem. 93, 71--78], it is possible to assess the evolutionary stability of this protein. There is little conservation of sequence in the N-terminal portion of the molecule (residues 1--26 numbering according to calf H2B), while the remainder of the protein, which we designate the C-terminal portion, is highly conserved. In the region of 27--125 residues, there are 9 substitutions in the composite data among the 98 positions, 8 of them conservative. These data indicate that very different selective pressures operate on the two different portions of the H2B molecule, implying the existence of two well-defined regions. Studies on the structure of the nucleosome by others have suggested that the C-terminal portion of H2B is involved in histone-histone interactions while the N-terminal portion is a relatively free "tail" binding to DNA. The sequence data indicate that the function of the C-terminal region of H2B requires considerable sequence specificity while that of the N-terminal region does not. 相似文献
10.
Expression of insulin metabolic effects can be obtained by anti-receptor antibodies without activation of the tyrosine kinase activity [O'Brien R. M., Soos M. A. and Siddle K. (1987) EMBO J. 6, 4003-4010; Forsayeth J. R., Caro J. F., Sinha M. K., Maddux B. A. and Goldfine I. D. (1987) Proc. natn. Acad. Sci. U.S.A. 84, 34,448-34,514; Ponzio G., Contreres J. O., Debant A., Baron V., Gautier N., Dolais-Kitabgi J. and Rossi B. (1988) EMBO J. 7, 4111-4117; Hawley D. M., Maddux B. A., Patel R. G., Wong K. Y., Mamula P. W., Firestone G. L., Brunetti A., Verspohl E. and Goldfine I. D. (1989) J. biol. Chem. 264, 2438-2444; Soos M. A., O'Brien R. M., Brindle N. P. J., Stigter J. M., Okamoto A. K., Whittaker J. and Siddle K. (1989) Proc. natn. Acad. Sci. U.S.A. 86, 5217-5221.]. Recently, we have proposed that receptor cross-linking is sufficient in itself to stimulate glycogen synthesis, even if aggregation was performed on receptors mutated on Tyr 1162 and Tyr 1163 and thus devoid of tyrosine kinase activity [Debant A., Ponzio G., Clauser E., Contreres J. O. and Rossi B. (1989) Biochemistry 28, 14-17]. The aim of this study was to gain information on the involvement of receptor clustering in the expression of the different insulin biological effects. To this end, we studied the mimetic effects of wheat-germ agglutinin, which is likely to induce receptor aggregation without interacting with the receptor protein moiety. Wheat-germ agglutinin failed to promote DNA synthesis, whereas the lectin behaved as a potent mimicker of insulin on tyrosine aminotransferase activity and amino-acid transport. However, this stimulatory effect did not parallel the activation of receptor autophosphorylation. Our data reinforce the idea that the expression of the metabolic effects of insulin are not strictly dependent on a general tyrosine kinase activation. 相似文献
11.
The complete amino acid sequence of a basic non-histone protein, H6, isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells, has been determined. Protein H6, first described by D. T. Wigle and G. H. Dixon [J. Biol. Chem. 246, 5636--5644 (1971)] was extracted with 5% trichloracetic acid and purified by ion-exchange chromatography on carboxymethyl-cellulose (CM-52). Sequence analysis was performed by automatic Edman degradation of the amino terminus of the intact protein and a series of large fragments derived by cleavage with chymotrypsin, staphylococcal protease and with mild acid to cleave at aspartic acid residues. Protein H6 possesses 69 residues and shows considerable similarities to the 89-residue calf thymus HMG-17 protein previously sequenced [Walker, J. M., Hastings, J. R. B. & Johns, E. W. (1977) Eur. J. Biochem. 76, 461--468]. B. Levy W. and G. H. Dixon [Proc. Natl Acad. Sci. U.S.A. 74, 2810--2814 (1977)] have shown that H6 is selectively solubilized when trout testis nuclei (or chromatin) are digested with DNase I under conditions which preferentially hydrolyze that portion of DNA enriched in transcribed sequences [Levy, W. B. & Dixon, G. H. (1977) Nucleic Acids Res. 4, 883--898]. Recently H6 has been located as a stoichiometric component of a distinct subset of trout testis nucleosomes that are complexed with a core nucleosome comprising 140 base pairs of DNA and the inner histones H2A, H2B, H3 and H4 [Levy, W. B., Connor, W. & Dixon, G. H. (1979) J. Biol. Chem., in the press]. 相似文献
12.
《Ibis》1989,131(1):155-161
B rickell , N. 1988. Ducks, Geese and Swans of Africa and its Outlying Islands.
B urr , E.W. (ed.) 1987. Companion Bird Medicine.
C lark , W.S. & W heeler , B.K. 1987.
C olston , P. & B urton , P. 1988.
C ramp , S. (ed.) 1988. Handbook of the Birds of Europe, the Middle East and North Africa: the Birds of the Western Palearctic, Vol. V.
H ill , D. & R obertson , P. 1988.
H ollom , P.A.D., P orter , R.F., C hristensen , S. & W illis , 1.1988. Birds of the Middle East and North Africa: A Companion Guide.
I lyichev , V.D. & Z ubakin , V.A. (eds) 1988. The Birds of the USSR: Lari. (Russian).
J ohnsgard , P. 1988. The Quails, Partridges and Francolins of the World.
P ätzold , R. 1987. Die Ohrenlerche.
P eck , G.K. & J ames , R.D. 1987. Breeding Birds of Ontario. Nidology and Distribution. Vol. 2: Passerines.
S eller , T.J. 1987. Bird Respiration. Vols 1 & 2.
W ells , R.G. & B elyavin , C.G. 1987. Egg Quality–Current Problems and Recent Advances. Poultry Science Symposium No. 20. 相似文献
B urr , E.W. (ed.) 1987. Companion Bird Medicine.
C lark , W.S. & W heeler , B.K. 1987.
C olston , P. & B urton , P. 1988.
C ramp , S. (ed.) 1988. Handbook of the Birds of Europe, the Middle East and North Africa: the Birds of the Western Palearctic, Vol. V.
H ill , D. & R obertson , P. 1988.
H ollom , P.A.D., P orter , R.F., C hristensen , S. & W illis , 1.1988. Birds of the Middle East and North Africa: A Companion Guide.
I lyichev , V.D. & Z ubakin , V.A. (eds) 1988. The Birds of the USSR: Lari. (Russian).
J ohnsgard , P. 1988. The Quails, Partridges and Francolins of the World.
P ätzold , R. 1987. Die Ohrenlerche.
P eck , G.K. & J ames , R.D. 1987. Breeding Birds of Ontario. Nidology and Distribution. Vol. 2: Passerines.
S eller , T.J. 1987. Bird Respiration. Vols 1 & 2.
W ells , R.G. & B elyavin , C.G. 1987. Egg Quality–Current Problems and Recent Advances. Poultry Science Symposium No. 20. 相似文献
13.
X-ray crystal structures have been determined for complexes of cytochrome P-450CAM with the substrates camphane, adamantane, and thiocamphor. Unlike the natural substrate camphor, which hydrogen bonds to Tyr96 and is metabolized to a single product, camphane, adamantane and thiocamphor do not hydrogen bond to the enzyme and all are hydroxylated at multiple positions. Evidently the lack of a substrate-enzyme hydrogen bond allows substrates greater mobility in the active site, explaining this lower regiospecificity of metabolism as well as the inability of these substrates to displace the distal ligand to the heme iron. Tyr96 is a ligand, via its carbonyl oxygen atom, to a cation that is thought to stabilize the camphor-P-450CAM complex [Poulos, T. L., Finzel, B. C., & Howard, A. J. (1987) J. Mol. Biol. 195, 687-700]. The occupancy and temperature factor of the cationic site are lower and higher, respectively, in the presence of the non-hydrogen-bonding substrates investigated here than in the presence of camphor, underscoring the relationship between cation and substrate binding. Thiocamphor gave the most unexpected orientation in the active site of any of the substrates we have investigated to date. The orientation of thiocamphor is quite different from that of camphor. That is, carbons 5 and 6, at which thiocamphor is primarily hydroxylated [Atkins, W. M., & Sligar, S. G. (1988) J. Biol. Chem. 263, 18842-18849], are positioned near Tyr96 rather than near the heme iron. Therefore, the crystallographically observed thiocamphor-P-450CAM structure may correspond to a nonproductive complex. Disordered solvent has been identified in the active site in the presence of uncoupling substrates that channel reducing equivalents away from substrate hydroxylation toward hydrogen peroxide and/or "excess" water production. A buried solvent molecule has also been identified, which may promote uncoupling by moving from an internal location to the active site in the presence of highly mobile substrates. 相似文献
14.
Kinetic and conformational effects of lysine substitutions for arginines 35 and 87 in the active site of staphylococcal nuclease 总被引:3,自引:0,他引:3
The high-resolution X-ray crystal structure of staphylococcal nuclease (SNase) suggests that the guanidinium groups of Arg 35 and Arg 87 participate as electrophilic catalysts in the attack of water on the substrate phosphodiester. Both arginine residues have been replaced with "conservative" lysine residues so that both the importance of these residues in catalysis and the effect of changes in electrostatic interactions on active site conformation can be assessed. The catalytic efficiencies of R35K and R87K are decreased by factors of 10(4) and 10(5) relative to wild-type SNase, with R87K showing a very significant reduction in its affinity for both DNA substrate and the competitive inhibitor thymidine 3',5'-bisphosphate (pdTp). The thermal denaturation behavior of both mutant enzymes differs from that of wild type both in the absence and in the presence of the active site ligands Ca2+ and pdTp. Both the 1H NMR chemical shifts and interresidue nuclear Overhauser effects (NOEs) of residues previously assigned to be in the hydrophobic core of SNase are altered in R35K and R87K. These observations, similar to those recently reported by our laboratories for substitutions for Glu 43 [Hibler, D. W., Stolowich, N. J., Reynolds, M. A., Gerlt, J. A., Wilde, J. A., & Bolton, P. H. (1987) Biochemistry 26, 6278; Wilde, J. A., Bolton, P. H., Dell'Acqua, M., Hibler, D. W., Pourmotabbed, T., & Gerlt, J. A. (1988) Biochemistry 27, 4127], suggest that lysine substitutions are not conservative in SNase and disrupt the conformation of the active site.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
S G Young F P Peralta B W Dubois L K Curtiss J K Boyles J L Witztum 《The Journal of biological chemistry》1987,262(34):16604-16611
In 1979, Steinberg and colleagues described a unique kindred with familial hypobetalipoproteinemia (Steinberg, D., Grundy, S. M., Mok, H. Y. I., Turner, J. D., Weinstein, D. B., Brown, W. V., and Albers, J. J. (1979) J. Clin. Invest. 64, 292-301). Recently, we demonstrated the existence of an abnormal species of apolipoprotein (apo-) B, apo-B37 (Mr = 203,000) in nine members of that kindred (Young, S. G., Bertics, S. J., Curtiss, L. K., and Witztum, J. L. (1987) J. Clin. Invest. 79, 1831-1841; Young, S. G., Bertics, S. J., Curtiss, L. K., Dubois, B. W., and Witztum, J. L. (1987) J. Clin. Invest. 79, 1842-1851). Apolipoprotein B37 contains only the amino-terminal portion of apo-B100. In affected individuals most of the apo-B37 is contained in the high density lipoprotein (HDL) fraction (d = 1.063-1.21 g/ml), where it is the principal apolipoprotein in a unique lipoprotein (Lp) particle, Lp-B37, which contains little, if any, apo-A-I. However, the most abundant lipoprotein in the HDL density fraction is a smaller particle, which contains apo-A-I, but no apo-B. The Lp-B37 particles were isolated from the HDL of affected individuals by immunoabsorption of apo-B37. Selected affinity antibodies specific for apo-B37 were used to prepare an anti-apo-B37-Sepharose 4B column. Lipoproteins not bound by the column (unbound HDL fraction) contained apo-A-I, but no apo-B. The Lp-B37, which was eluted from the column with 3 M KI, contained apo-B37 and trace amounts of apo-A-I, but no apo-B100. Over a 4-h period, normal human fibroblasts degraded 10-fold more 125I-low density lipoprotein (LDL) than 125I-Lp-B37. Also, whereas addition of excess unlabeled LDL markedly reduced degradation of 125I-LDL, it did not significantly reduce the degradation of 125I-Lp-B37. Unlabeled Lp-B37 did not inhibit uptake and degradation of 125I-LDL by fibroblasts. These data suggest that the amino-terminal portion of apo-B100, when expressed on a naturally occurring lipoprotein particle, does not contain a functional apo-B,E(LDL) receptor binding domain. 相似文献
16.
17.
P Caffrey B Green L C Packman B J Rawlings J Staunton P F Leadlay 《European journal of biochemistry》1991,195(3):823-830
The C-terminal region of a multifunctional polypeptide from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea is predicted to contain an acyl carrier protein and a thioesterase or acyltransferase activity [Cortes, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J. & Leadlay, P. F. (1990) Nature 348, 176-178]. Site-directed mutagenesis by means of the polymerase chain reaction was used to construct an efficient pT7-based expression plasmid for this domain. The recently developed technique of electrospray mass spectrometry was used to demonstrate that the purified protein had not been post-translationally modified by attachment of a 4'-phosphopantetheine group. However, treatment with the serine proteinase inhibitor phenylmethylsulphonyl fluoride led to highly selective labelling of the predicted active site of the thioesterase or acyltransferase. 相似文献
18.
Pig kidney medium-chain acyl-CoA dehydrogenase is specifically alkylated at a methionine residue by treatment with iodoacetate at pH 6.6. This residue corresponds to Met249 in the human medium-chain acyl-CoA dehydrogenase sequence [Kelly, D. P., Kim, J. J., Billadello, J. J., Hainline, B. E., Chu, T. W., & Strauss, A. W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4068-4072]. The S-carboxymethylated dehydrogenase shows a drastically lowered affinity for octanoyl-CoA (from submicromolar to 65 microM), but retains about 23% of the maximal activity of the native enzyme. In addition, alkylation perturbs the internal redox equilibrium: E.FADox.octanoyl-CoA K2 in equilibrium with E.FAD2e.octenoyl-CoA K2 ranges from about 9 for the native enzyme to about 0.2 for the homogeneously modified protein. This effect is not due to a significant change in the redox potential of the free enzyme upon alkylation. Rather, carboxymethylation weakens the preferential binding of enoyl-CoA product to the reduced enzyme (K3) compared to octanoyl-CoA binding to the oxidized dehydrogenase (K1) that is required to pull the substrate thermodynamically uphill. Thus, the ratio of dissociation constants, K1/K3, decreases from about 15,000 for the native enzyme to only 330 upon carboxymethylation of Met249. Binding studies with a variety of acyl-CoA analogues and manipulation of enzyme redox potentials by substitution of the natural prosthetic group by 8-Cl-FAD confirm the thermodynamic effects of alkylation. 相似文献
19.
This work broadens the scope and improves the time resolution of spin-probe oximetry, a technique in which small nitroxide spin probes detect oxygen consumption via change in their relaxation properties [Froncisz, W., Lai, C.-S., & Hyde, J. S. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 411-415]. For rapid oxygen kinetic studies we combined the methodology of spin-probe oximetry with a recently developed loop-gap resonator, stopped-flow EPR system [Hubbell, W. L., Froncisz, W., & Hyde, J. S. (1987) Rev. Sci. Instrum. 58, 1879-1886]. The technique used microliter volumes of reactant solutions. Enzymatic consumption of oxygen by cytochrome c oxidase in the presence of ferrocytochrome c substrate was followed continuously in time under limited-turnover conditions, where the concentration of oxygen consumed often was comparable to or less than the amount of enzyme present. In detecting less than micromolar oxygen concentration changes, we have achieved a time resolution of the order 30 ms when flow is stopped. Oxygen consumption was followed under two different limited-turnover conditions: In the first, the amount of oxygen consumed was limited by available ferrocytochrome c, and the time course of oxygen consumption and its pH dependence were compared with the optically detected ferrocytochrome c consumption. In the second, the oxygen consumed was ultimately limited by the availability of oxygen itself while ferrocytochrome c was regenerated and remained in excess.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
Bernocco S Steiglitz BM Svergun DI Petoukhov MV Ruggiero F Ricard-Blum S Ebel C Geourjon C Deleage G Font B Eichenberger D Greenspan DS Hulmes DJ 《The Journal of biological chemistry》2003,278(9):7199-7205
Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that can stimulate the action of tolloid metalloproteinases, such as bone morphogenetic protein-1, on a procollagen substrate, by up to 20-fold. The PCPE molecule consists of two CUB domains followed by a C-terminal NTR (netrin-like) domain. In order to obtain structural insights into the function of PCPE, the recombinant protein was characterized by a range of biophysical techniques, including analytical ultracentrifugation, transmission electron microscopy, and small angle x-ray scattering. All three approaches showed PCPE to be a rod-like molecule, with a length of approximately 150 A. Homology modeling of both CUB domains and the NTR domain was consistent with the low-resolution structure of PCPE deduced from the small angle x-ray scattering data. Comparison with the low-resolution structure of the procollagen C-terminal region supports a recently proposed model (Ricard-Blum, S., Bernocco, S., Font, B., Moali, C., Eichenberger, D., Farjanel, J., Burchardt, E. R., van der Rest, M., Kessler, E., and Hulmes, D. J. S. (2002) J. Biol. Chem. 277, 33864-33869) for the mechanism of action of PCPE. 相似文献