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1.
B J Marsden F D Sauer B A Blackwell J K Kramer 《Biochemical and biophysical research communications》1989,159(3):1404-1410
The methylcoenzyme M methylreductase reaction has an absolute requirement for 7-mercaptoheptanoylthreonine phosphate or component B, which is the active component of the intact molecule previously referred to as cytoplasmic cofactor. A hydrolytic fragment of cytoplasmic cofactor has been purified and identified as uridine 5'-(O-2-acetamido-2-deoxy-beta-manno-pyranuronosyl acid (1----4)-2-acetamido-2-deoxy-alpha-glucopyranosyl diphosphate) by high resolution NMR and fast atom bombardment mass spectro-metry. It is postulated that UDP-disaccharide may function to anchor 7-mercaptoheptanoyl threonine phosphate at the active site of the methyl-reductase enzyme complex. 相似文献
2.
The role of 7-mercaptoheptanoylthreonine phosphate in the methylcoenzyme M methylreductase system from Methanobacterium thermoautotrophicum 总被引:4,自引:0,他引:4
The structure of component B of the methylcoenzyme M methylreductase system from Methanobacterium thermoautotrophicum was recently found to be 7-mercaptoheptanoylthreonine phosphate (HS-HTP). Three potential roles for this cofactor were considered. First, a methyl thioether derivative of the cofactor was synthesized to investigate its possible role as a methyl donor. This derivative was found to be incapable of acting as a substrate for methanogenesis and proved inhibitory. Secondly, an adenylated form of the cofactor was considered as the potential active form of the coenzyme. This possibility was ruled out based upon collaborative observations with Ankel-Fuchs et al. (FEBS Lett., in press) that HS-HTP is required by the methylreductase system even when ATP is not. Finally, HS-HTP was found to act as a reductant in a partially-purified methylreductase preparation that was incubated under nitrogen. The rate of methane production from HS-HTP exceeded that from other thiols or hydrogen. 相似文献
3.
Component C of the methylcoenzyme M methylreductase system contains bound 7-mercaptoheptanoylthreonine phosphate (HS-HTP) 总被引:5,自引:0,他引:5
The structure of component B of the methylcoenzyme M methylreductase system of Methanobacterium thermoautotrophicum was recently found to be 7-mercaptoheptanoylthreonine phosphate (HS-HTP). The work described here demonstrates that this compound is found in two forms: enzyme-free and enzyme-bound. HS-HTP was found to be bound to component C of the methylcoenzyme M methylreductase system. The cofactor extracted from the protein by heat denaturation was found to comigrate with the mixed disulfide of HS-HTP and 2-mercaptoethanol by high-performance liquid chromatography, suggesting HS-HTP is not modified in the bound state. 相似文献
4.
J L Johnson L W Indermaur K V Rajagopalan 《The Journal of biological chemistry》1991,266(19):12140-12145
The chlorate-resistant mutants of Escherichia coli are affected in the biosynthesis of the molybdenum cofactor and show pleiotropic loss of the activities of those enzymes which require the cofactor. The molybdenum cofactor in all molybdoenzymes other than nitrogenase is a complex of the metal with a unique pterin termed molybdopterin. The molybdenum cofactor in a number of E. coli enzymes has been shown to contain GMP in addition to the metal-molybdopterin complex, with the GMP appended in pyrophosphate linkage to the terminal phosphate ester on the molybdopterin side chain. In this paper, we have examined the biochemistry of the chlB mutant and show that the gene product of the chlB locus is essential for the addition of the GMP moiety to form molybdopterin guanine dinucleotide, a step which occurs late in the cofactor biosynthetic pathway in E. coli. Sensitive techniques were developed for the identification of fluorescent derivatives of molybdopterin and of molybdopterin guanine dinucleotide in extracts of E. coli cells. Wild type cells were shown to contain both molybdopterin and molybdopterin guanine dinucleotide, while cells of chlB mutants were found to contain elevated levels of molybdopterin but no detectable molybdopterin guanine dinucleotide. 相似文献
5.
Reductive activation of the methyl coenzyme M methylreductase system of Methanobacterium thermoautotrophicum delta H. 总被引:11,自引:8,他引:3
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When titanium(III) citrate was used as electron donor for the reduction of methyl coenzyme M by the methyl coenzyme M methylreductase system of Methanobacterium thermoautotrophicum delta H, component A1 was no longer required. The simpler system thus obtained required components A2, A3, and C as well as catalytic amounts of ATP, vitamin B12, and the disulfide of 7-mercaptoheptanoylthreonine phosphate in addition to titanium(III) citrate. This three component enzyme system also could produce CH4 when stoichiometric amounts of 7-mercaptoheptanoylthreonine phosphate were used as a source of electrons under an H2 atmosphere. When 7-mercaptoheptanoylthreonine phosphate or H2 was used alone no CH4 was produced, indicating a dual requirement for reducing equivalents: one to activate the methylreductase system and the other to reduce methyl coenzyme M. This is the first evidence that the activation of methyl coenzyme M methylreductase is a reductive process. 相似文献
6.
Disrupted potato starch granules obtained in the presence of 8 M urea were shown to increase [14C] glucose incorporation from labeled ADP-glucose or UDP-glucose into starch, as compared to intact grains. Labeled glucose or maltose units were found to be incorporated through a linkage that produced cyclic phosphate esters upon mild alkaline treatment and was sensitive to hydrolysis at pH 2.0. Both properties of this linkage strongly resembled those of the pyrophosphate bond of ADP-glucose or UDP-glucose. 相似文献
7.
Ciszak EM Korotchkina LG Dominiak PM Sidhu S Patel MS 《The Journal of biological chemistry》2003,278(23):21240-21246
The derivative of vitamin B1, thiamin pyrophosphate, is a cofactor of enzymes performing catalysis in pathways of energy production. In alpha2beta2-heterotetrameric human pyruvate dehydrogenase, this cofactor is used to cleave the Calpha-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites has not yet been understood. To understand the mechanism of action of this enzyme, we determined the crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95-A resolution. We propose a model for the flip-flop action of this enzyme through a concerted approximately 2-A shuttle-like motion of its heterodimers. Similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase with functionally related enzymes suggests that this newly defined shuttle-like motion of domains is common to the family of thiamin pyrophosphate-dependent enzymes. 相似文献
8.
G.Ya. Sheflyan E.A. Kubareva S.A. Kuznetsova A.S. Karyagina I.I. Nikolskaya E.S. Gromova Z.A. Shabarova 《FEBS letters》1996,390(3):307-310
Specific and non-specific interactions SsoII restriction endonuclease (R·SsoII) were probed by the method of covalent attachment to modified DNA containing an active monosubstituted pyrophosphate internucleotide bond instead of a phosphodiester one. R·SsoII with six N-terminal His residues was shown to be cross-linked to duplexes with this type of modification, either containing or not the recognition sequence. Competition experiments with covalent attachment of R·SsoII to activated DNAs demonstrated the similar affinity of the enzyme to cognate and non-cognate DNAs in the absence of cofactor, Mg2+ ions. 相似文献
9.
A member of a new class of GTP cyclohydrolases produces formylaminopyrimidine nucleotide monophosphates 总被引:3,自引:0,他引:3
The hyperthermophilic euryarchaeon Methanococcus jannaschii has no recognizable homologues of the canonical GTP cyclohydrolase enzymes that are required for riboflavin and pteridine biosyntheses. Instead, it uses a new type of thermostable GTP cyclohydrolase enzyme that produces 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone ribonucleotide monophosphate and inorganic phosphate. Whereas canonical GTP cyclohydrolases produce this formylamino-pyrimidine nucleotide as a reaction intermediate, this compound is shown to be an end product of the purified recombinant M.jannaschii enzyme. Unlike other enzymes that hydrolyze the alpha-beta phosphate anhydride bond of GTP, this new enzyme completely hydrolyzes pyrophosphate to inorganic phosphate. As a result, the enzyme has a steady-state turnover of 21 min(-)(1), which is much faster than those of canonical GTP cyclohydrolase enzymes. The effects of substrate analogues and inhibitors suggest that the GTP cyclohydrolase and pyrophosphate phosphohydrolase activities occur at independent sites, although both activities depend on Mg(2+). 相似文献
10.
P1-Dolichyl P2-ALPHA-D-mannopyranosyl pyrophosphate (9) has been chemically synthesized by a method developed for the corresponding citronellyl derivative, which also contains a saturated alpha isoprene residue. In each case, the P1-polyisoprenyl P2-diphenyl pyrophosphate was treated with 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate to give a fully acetylated pyrophosphate diester, which was purified chromatographically and subsequently deacetylated. The citronellyl and dolichyl pyrophosphate diesters were compared with the previously synthesized citronellyl and dolichyl alpha-D-mannopyranosyl phosphate, respectively, by chromatography and by hydrolysis experiments. Good separations of the monophosphate from the corresponding pyrophosphate were achieved by silica gel tlc in a variety of solvent systems. Brief dilute acid hydrolysis of both the mono- and pyrophosphate diesters gave D-mannose and no alpha-D-mannosyl phosphate, the other products being polyprenyl phosphate and pyrophosphate, respectively. When the polyprenyl alpha-D-mannopyranosyl mono- and pyrophosphate diesters were treated with hot dilute alkali, the major products were polyprenyl phosphate and substances arising from the breakdown of D-mannose, indicating that the alpha-D-mannosyl phosphate bond was the most labile linkage in both compounds. However, the formation of a small proportion of free dolichol indicated that alpha-D-mannosyl phosphate was also formed to a minor extent. The interpretation of the results of the alkaline hydrolysis was complicated by the instability of D-mannose under basic conditions, it being almost completely degraded by even a brief treatment. 相似文献
11.
The crystal structure of Bacillus subtilis thiamin phosphate synthase complexed with the reaction products thiamin phosphate and pyrophosphate has been determined by multiwavelength anomalous diffraction phasing techniques and refined to 1.25 A resolution. Thiamin phosphate synthase is an alpha/beta protein with a triosephosphate isomerase fold. The active site is in a pocket formed primarily by the loop regions, residues 59-67 (A loop, joining alpha3 and beta2), residues 109-114 (B loop, joining alpha5 and beta4), and residues 151-168 (C loop, joining alpha7 and beta6). The high-resolution structure of thiamin phosphate synthase complexed with its reaction products described here provides a detailed picture of the catalytically important interactions between the enzyme and the substrates. The structure and other mechanistic studies are consistent with a reaction mechanism involving the ionization of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate at the active site to give the pyrimidine carbocation. Trapping of the carbocation by the thiazole followed by product dissociation completes the reaction. The ionization step is catalyzed by orienting the C-O bond perpendicular to the plane of the pyrimidine, by hydrogen bonding between the C4' amino group and one of the terminal oxygen atoms of the pyrophosphate, and by extensive hydrogen bonding and electrostatic interactions between the pyrophosphate and the enzyme. 相似文献
12.
Mechanism of action of a yeast RNA ligase in tRNA splicing 总被引:44,自引:0,他引:44
The yeast endonuclease and ligase activities that carry out the splicing of tRNA precursors in vitro have been physically separated. The properties of a partially purified ligase fraction were examined. The ligase requires a divalent cation and a nucleoside triphosphate as cofactor. The product of ligation is a 2′-phosphomonoester, 3′,5′-phosphodiester linkage. The phosphate in the newly formed phosphodiester bond comes from the γ position of ATP, while the 2′ phosphate is derived from the RNA substrate. An adenylylated enzyme intermediate was identified by incorporation of label from α-32P-ATP. Adenylylation was reversed by pyrophosphate, releasing ATP, whereas ligation was accompanied by release of AMP. Polynucleotide kinase and cyclic phosphodiesterase activities copurify with the adenylylated protein and may be required for the tRNA splicing reaction. 相似文献
13.
Yeast inorganic pyrophosphatase is a dimer of identical subunits. Previous work (Rapoport, T.A., et al. (1973) Eur. J. Biochem. 33, 341) indicated the presence of two different Mn2+ binding sites per subunit. In the present work, the binding of inorganic phosphate to the Mn2+-inorganic pyrophosphatase complex has been studied by 1H and 31P nuclear magnetic resonance. Two distinct phosphate sites have been found, having dissociation constants of 0.24 mM and 18 mM. The Mn2+-31P distance from tightly bound Mn2+ to phosphate bound in the low affinity site (6.2 A) is consistent with outer sphere binding. Binding to both phosphate sites can be simultaneously inhibited by the pyrophosphate analogue, hydroxymethanebisphosphonate, providing evidence for the physical proximity of these two sites. The weaker Mn2+ site is apparently far from both phosphate sites. From the magnitudes of the dissociation constants found for both phosphate and analogue binding and the recent work of P.D. Boyer and his co-workers (private communication) on enzyme-catalyzed phosphate-water exchange, it appears unlikely that the hydrolysis of enzyme-bound pyrophosphate is the rate-determining step in the overall enzymatic catalysis of pyrophosphate hydrolysis, at least when Mn2+ is the required divalent metal ion cofactor. 相似文献
14.
Chemical reactions in double-stranded nucleic acids. IX. Directed introduction of substituted pyrophosphate bonds into DNA structure 总被引:2,自引:0,他引:2
An effective synthesis of oligodeoxyribonucleotides containing a substituted pyrophosphate bond in the definite position of the sugar-phosphate backbone has been developed by template-directed condensation of two heptanucleotides. One of them containing 5'-phosphate group to be activated and 3'-phosphate group of the other being substituted with ethoxy-, buthylamino-, morpholino- or ethyl glycinate residues. Water-soluble carbodiimide (EDAC) proved to be more efficient in the phosphate group activation than N-hydroxybenzotriazole ester (yields of substituted pyrophosphates 35-80 and 10-15% respectively). The substituted pyrophosphate bong is quite stable in neutral aqueous solution. Mild conditions of selective cleavage of this bond yielding the initial oligonucleotides were found. 相似文献
15.
Characterization of the Saccharomyces cerevisiae cis-prenyltransferase required for dolichyl phosphate biosynthesis 总被引:1,自引:0,他引:1
The prenyltransferase involved in the biosynthesis of dolichyl phosphate has been characterized in Saccharomyces cerevisiae. Although the enzyme is predominantly membrane-bound, a significant percentage was found in the soluble fraction. The prenyltransferase preferentially utilizes farnesyl pyrophosphate as the allylic substrate and isopentenyl pyrophosphate as cosubstrate with half-maximal velocities obtained at 25 and 6.7 microM, respectively. The enzymatic activity is sensitive to sulfhydryl reagents and is inhibited by all detergents tested, except 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate at concentrations less than 5 mM. The product of the reaction has been characterized as an alpha-unsaturated polyprenyl pyrophosphate, containing 12-15 isoprene units, approximately two isoprene units shorter than the endogenous yeast dolichyl phosphate. The stereochemistry of addition of isoprene units by the prenyltransferase was shown to be cis by a comparison of the HPLC retention time for a pentadecaprenyl phosphate derived from the in vitro reaction product with that for an authentic mixture of alpha-cis- and alpha-trans-pentadecaprenyl phosphates. 相似文献
16.
A novel biosynthesis of medium chain length alpha-ketodicarboxylic acids in methanogenic archaebacteria 总被引:1,自引:0,他引:1
R H White 《Archives of biochemistry and biophysics》1989,270(2):691-697
Gas chromatographic-mass spectrometric analysis on the distribution of alpha-ketodicarboxylic acids in various bacteria determined that alpha-ketoglutarate and alpha-ketoadipate are widely distributed in all the bacteria examined, whereas alpha-ketopimelate and alpha-ketosuberate are found only in the methanogenic archaebacteria. Labeling experiments with stable isotopes indicated that each of these acids arises from alpha-ketoglutarate by repeated alpha-ketoacid chain elongation. The final product in this series of reactions, alpha-ketosuberate, serves in the methanogenic bacteria as the biosynthetic precursor to the 7-mercaptoheptanoic acid portion of 7-mercaptoheptanoylthreonine phosphate, a methanogenic coenzyme. 相似文献
17.
Dean J. Danner Sandra K. Lemmon Louis J. Elsas 《Archives of biochemistry and biophysics》1980,202(1):23-28
Thiamin pyrophosphate, CoASH, and NAD+ have been shown to reversibly bind to the purified bovine liver mitochondrial branched-chain α-ketoacid dehydrogenase complex. When saturated with thiamin pyrophosphate, the complex was more stable to heat and chymotrypsin inactivation. Under identical saturating conditions a conformational change in the complex was observed by circular dichroism spectroscopy. We postulate that thiamin pyrophosphate can increase the biological half-life of the in vivo, membrane-bound complex through conformational changes induced by the binding of this cofactor. 相似文献
18.
The effect of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC) on the reactions catalyzed by the glucose-6-phosphatase system of rat liver microsomes was studied. Modification of the intact microsomes by CMC leads to the inhibition of the glucose-6-phosphatase, pyrophosphate:glucose and carbamoyl-phosphate : glucose phosphotransferase activities of the system. The activities are restored by the disruption of the microsomal permeability barrier. The mannose-6-phosphate, pyrophosphate, and carbamoyl-phosphate phosphohydrolase activities of the intact as well as the disrupted microsomes were not affected by CMC. It follows from the results obtained that CMC inactivates the microsomal glucose-6-phosphate translocase, the inactivation is a result of the modification of a single sulfhydryl or amino group of the translocase; pyrophosphate, carbamoyl phosphate and inorganic phosphate are transported across the microsomal membrane without participation of the glucose-6-phosphate translocase; pyrophosphate and carbamoyl phosphate may act as the phosphate donors in the glucose phosphorylation reactions in vivo. 相似文献
19.
Two enantiomeric analogues of farnesyl pyrophosphate (1) were tested as inhibitors and anomalous substrates of trichodiene synthase, which catalyzes the cyclization of trans,trans-farnesyl pyrophosphate (1) to the sesquiterpene hydrocarbon trichodiene (2). The reaction has been shown to involve preliminary isomerization of 1 to the tertiary allylic isomer nerolidyl pyrophosphate (3) which is cyclized without detectable release of the intermediate from the active site of the cyclase. Both (7S)-trans-6,7-dihydrofarnesyl pyrophosphate (7a) and (7R)-trans-6,7-dihydrofarnesyl pyrophosphate (7b), prepared from (3R)- and (3S)- citronellol (9a and 9b), respectively, proved to be modest competitive inhibitors of trichodiene synthase. The values of Ki(7a), 395 nM, and Ki(7b), 220 nM, were 10-15 times the observed Km for 1 and half the Ki of inorganic pyrophosphate alone. Incubation of either 7a or 7b with trichodiene synthase resulted in formation of a mixture of products which by radio/gas-liquid chromatographic and GC/selected ion mass spectrometric analysis was shown to be composed of 80-85% isomeric trienes 19-21 and 15-20% allylic alcohols 12 and 18. Examination of the water-soluble products resulting from incubation of 7a also revealed the generation of 24% of the isomeric cis-6,7-dihydrofarnesyl pyrophosphate (26). The combined rate of formation of anomalous alcoholic and olefinic products was 10% the Vmax determined for the conversion of 1 to 2. The results can be explained by initial enzyme-catalyzed isomerization of dihydrofarnesyl pyrophosphate (7) to the corresponding tertiary allylic isomer dihydronerolidyl pyrophosphate (8). Since the latter intermediate is unable to cyclize due to the absence of the 6,7-double bond, ionization of 8 and quenching of the resulting ion pair by deprotonation, capture of water, or collapse to the isomeric primary pyrophosphate esters will generate the observed spectrum of anomalous products. 相似文献
20.
Abstract Extracts of acetate-grown Methanosarcina barkeri strain Fusaro formed methane from acetate plus ATP and form acetyl phosphate under H2 . Coenzyme A (CoA) is stimulatory. Inhibitors of methanogenesis are cyanide, propyliodide and bromoethanesulfonic acid. In cofactor-free extracts methanogenic activity from acetate was restored by addition of ATP, CoA, coenzyme M and 7-mercaptoheptanoylthreonine phosphate.
An enzyme-bound corrinoid was found to be involved in methanogenesis from acetate. 相似文献
An enzyme-bound corrinoid was found to be involved in methanogenesis from acetate. 相似文献