Abstract:
:The self-splicing ai5g group II intron was transformed into a three-part ribozyme that site-specifically cleaves small oligonucleotide substrates with multiple turnover. The ribozyme is composed of intron domain 1 (D1, 425 nucleotides), with catalytically essential domain 5 (D5, 58 nucleotides) provided separately as a reaction cofactor. Together, the D1/D5 complex cleaves small substrates analogous in sequence to the 5'-splice site of the intron. Activity of the ribozyme was studied using a combination of single- and multiple-turnover experiments in which the concentrations of the RNA components were varied in order to probe their individual role in the overall mechanism. Values for kcat, Km, and kcat/Km were the same within experimental error for the two enzymological approaches. These kinetic analyses reveal that the ribozyme utilizes a classic Michaelis-Menten reaction mechanism in which the chemical step of catalysis (kcat = kchem = approximately 0.03 min-1 at full saturation) is rate limiting for the overall reaction. The D1/D5 complex binds tightly to the substrate (Km = 6.3 nM) and specifically recognizes sequences both 5' and 3' to the ribozyme cleavage site. These studies represent the first quantitative analysis of group II recognition and affinity for the 5'-splice site. As observed in previous studies on the role of D5 RNA, D5 binds tightly to the ternary complex (Km = 870 nM). The second-order rate constant for RNA cleavage (kcat/Km = 3.3 x 10(6)) is an order of magnitude slower than that observed for other ribozymes in this mechanistic class, all of which are rate-limited by steps other than chemistry. That kcat equals kchem in this ribozyme is supported by the overall kinetic analysis and by the observation that an Rp phosphorothioate is cleaved approximately 3-fold more slowly than a phosphate at the cleavage site. These studies represent a preliminary examination of stereochemical preference by a group II intron active site in the transition state. The substrate specificity, reaction conditions, and mutational sensitivity of this ribozyme are consistent with a reaction analogous to the first step of group II intron self-splicing, although its stereochemical preference is analogous to a second-step reversal.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Michels WJ Jr,Pyle AMdoi
10.1021/bi00009a028subject
Has Abstractpub_date
1995-03-07 00:00:00pages
2965-77issue
9eissn
0006-2960issn
1520-4995journal_volume
34pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::Peptidylglycine monooxygenase (PHM) catalyzes the final step in the biosynthesis of amidated peptides that serve as important signaling molecules in numerous endocrine pathways. The catalytic mechanism has attracted much attention because of a number of unique attributes, including the presence of a pair of uncoupled ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi4015264
更新日期:2014-02-18 00:00:00
abstract::The folding of many globular proteins from the unfolded (U) to the native (N) state appears to be describable by a two-state N ↔ U model, which has led to the general belief that protein folding occurs in a highly cooperative manner. One reason for the widespread belief in "two-state folding" is that protein folding r...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.8b00698
更新日期:2018-10-09 00:00:00
abstract::The heme binding sites of rabbit histidine-rich glycoprotein (HRG), 94 kDa, were studied with rose bengal (RB), a fluorescein derivative that associates with histidine residues. Difference absorbance spectra indicate that HRG binds RB at two thermodynamically preferred sites (Kd approximately 2 microM) that are spectr...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00342a034
更新日期:1985-10-08 00:00:00
abstract::Double-stranded complementary deoxyribonucleic acid (cDNA) transcribed in vitro from a partially pure myosin light-chain messenger ribonucleic acid (mRNA) of the chick embryonic heart was cloned in Escherichia coli strain chi 1776 by using the HindIII cleavage site in the plasmid pBR322. The insertion of essentially f...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00592a019
更新日期:1979-12-11 00:00:00
abstract::Defects in the MMACHC gene represent the most common disorder of cobalamin (Cbl) metabolism, affecting synthesis of the enzyme cofactors adenosyl-Cbl and methyl-Cbl. The encoded MMACHC protein binds intracellular Cbl derivatives with different upper axial ligands and exhibits flavin mononucleotide (FMN)-dependent decy...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi300150y
更新日期:2012-06-26 00:00:00
abstract::Deazapurine-containing secondary metabolites comprise a broad range of structurally diverse nucleoside analogues found throughout biology, including various antibiotics produced by species of Streptomyces bacteria and the hypermodified tRNA bases queuosine and archaeosine. Despite early interest in deazapurines as ant...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi900400e
更新日期:2009-05-12 00:00:00
abstract::P-450(15)alpha is a form of cytochrome P-450 purified from liver microsomes of female 129/J mice that is specific for oxidation of testosterone to its 15 alpha-hydroxylated product. Testosterone 15 alpha-hydroxylase activity that was inhibited by anti-P-450(15)alpha antibody was approximately 50 times higher in renal ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00365a028
更新日期:1986-08-26 00:00:00
abstract::The mechanism of inactivation of human leukocyte elastase (HLE) by the chloromethyl ketone MeOSuc-Ala-Ala-Pro-Val-CH2Cl was investigated. The dependence of the first-order rate constant for inactivation on concentration of chloromethyl ketone is hyperbolic and suggests formation of a reversible "Michaelis complex" pri...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00367a011
更新日期:1986-09-23 00:00:00
abstract::Utilizing structure-based design, we have previously demonstrated that it is possible to obtain selective inhibitors of protein-tyrosine phosphatase 1B (PTP1B). A basic nitrogen was introduced into a general PTP inhibitor to form a salt bridge to Asp48 in PTP1B and simultaneously cause repulsion in PTPs containing an ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi011389l
更新日期:2001-12-11 00:00:00
abstract::Human alkyladenine DNA glycosylase (AAG) locates and excises a wide variety of structurally diverse alkylated and oxidized purine lesions from DNA to initiate the base excision repair pathway. Recognition of a base lesion requires flipping of the damaged nucleotide into a relatively open active site pocket between two...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi101856a
更新日期:2011-03-22 00:00:00
abstract::Terpene synthases are potentially useful biocatalysts for the synthesis of valuable compounds, such as anticancer drugs and antibiotics. The design of altered activities requires better knowledge of their mechanisms, for example, an understanding of the complex conformational changes that are part of their catalytic c...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi400898k
更新日期:2013-11-12 00:00:00
abstract::The key polyamine catabolizing enzyme spermidine-spermine N1-acetyltransferase (SSAT) is among the few genes known to be inducible by the natural polyamines. Certain polyamine analogs markedly exaggerate this response and thus provide useful tools for studying the underlying regulatory mechanisms. As shown here, the a...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9612273
更新日期:1996-11-12 00:00:00
abstract::Neutral oligosaccharides, which accounted for 74% of the total N-linked sugar chains released by hydrazinolysis of rat small intestinal aminopeptidase N, were investigated on a structural basis. They are mainly composed of complex-type sugar chains with tri- and tetraantennary structures, and small amounts of high man...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00101a028
更新日期:1991-09-17 00:00:00
abstract::Sulfonylureas and glinides are commonly used oral insulin secretagogues (ISs) that act on the pancreatic ATP-sensitive potassium (KATP) channel to promote insulin secretion in order to lower the blood glucose level. Physiologically, KATP channels are inhibited by intracellular ATP and activated by Mg-ADP. Therefore, t...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.9b00692
更新日期:2020-01-14 00:00:00
abstract::Physicochemical properties of recombinant human erythropoietin were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human urine when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Sedimenta...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00383a034
更新日期:1987-05-05 00:00:00
abstract::The proton nuclear magnetic resonance spectrum of human adult deoxyhemoglobin in D2O in the region from 6 to 20 ppm downfield from the proton resonance of residual water shows a number of hyperfine shifted proton resonances that are due to groups on or near the alpha and beta hemes. The sensitivity of these resonances...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00590a031
更新日期:1979-11-13 00:00:00
abstract::Lactoferrin (Lf) and serum transferrin (Tf) combine high-affinity iron binding with an ability to release this iron at reduced pH. Lf, however, retains iron to significantly lower pH than Tf, giving the two proteins distinct functional roles. In this paper, we compared the iron-release profiles for human Lf, Tf, and t...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0001224
更新日期:2000-06-06 00:00:00
abstract::A compound tentatively identified as O2-methyl-5-carboxymethyluridine (cm5Um) was recently isolated in this laboratory from bulk yeast transfer RNA (Gray, M. W. (1975), Can, J. Biochem. 53, 735-746). Alkaline hydrolysis of yeast tRNA releases this nucleoside as part of an alkali-stable dinucleotide, cm5Um-Ap, from whi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00659a017
更新日期:1976-07-13 00:00:00
abstract::The transmission of regulatory signals between the alpha- and beta-subunits of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium has been investigated by monitoring the luminescence properties of the enzyme in the presence and in the absence of the alpha-subunit ligand DL-alpha-glycerol 3-phos...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00148a014
更新日期:1992-08-25 00:00:00
abstract::The reaction of ferric cytochrome c peroxidase (CcP) from Saccharomyces cerevisiae with peroxide produces compound I, characterized by both an oxyferryl iron center and a protein-based free radical. The electron paramagnetic resonance (EPR) signal of the CcP compound I radical can be resolved into a broad majority com...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00221a036
更新日期:1991-02-19 00:00:00
abstract::The inactivation of excited rhodopsin in the presence of ATP, rhodopsin kinase, and/or arrestin has been studied from its effect on the two subsequent steps in the light-induced enzymatic cascade: metarhodopsin II catalyzed activation of G-protein and G-protein-dependent activation of cGMP phosphodiesterase. The inact...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00405a049
更新日期:1988-03-08 00:00:00
abstract::The role of bacterial DnaJ protein as a cochaperone of DnaK is strongly appreciated. Although DnaJ unaccompanied by DnaK can bind unfolded as well as native substrate proteins, its role as an individual chaperone remains elusive. In this study, we demonstrate that DnaJ binds a model non-native substrate with a low nan...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi301543g
更新日期:2013-02-12 00:00:00
abstract::The C2A domain of synaptotagmin 7 (Syt7) is a Ca(2+) and membrane binding module that docks and inserts into cellular membranes in response to elevated intracellular Ca(2+) concentrations. Like other C2 domains, Syt7 C2A binds Ca(2+) and membranes primarily through three loop regions; however, it docks at Ca(2+) conce...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.5b00422
更新日期:2015-09-22 00:00:00
abstract::The signaling apparatus that controls bacterial chemotaxis is composed of a core complex containing chemoreceptors, the histidine autokinase CheA, and the coupling protein CheW. Site-specific spin labeling and pulsed dipolar ESR spectroscopy (PDS) have been applied to investigate the structure of a soluble ternary com...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi100055m
更新日期:2010-05-11 00:00:00
abstract::Eubacterial peptidyl-tRNA hydrolase (PTH) recycles all N-blocked aminoacyl-tRNA molecules but initiator formyl-methionyl-tRNAfMet, the acceptor helix of which is characterized by a 1-72 mismatch. Positive selection by PTH of noninitiator tRNA molecules with a full 1-72 base pair is abolished, however, upon the removal...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi982657r
更新日期:1999-04-20 00:00:00
abstract::Thyroxine and analogues inhibit rat liver aminoacyl-tRNA synthetase activity for phenylalanine and tyrosine. A high yield purification of the major cytoplasmic form of phenylalanyl-tRNA synthetase (C1) and its characterization is reported. Polyribosome-bound and other sedimentable forms are found to be indistinguishab...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00647a017
更新日期:1976-01-27 00:00:00
abstract::Tubulin polymerization promoting proteins (TPPPs) constitute a eukaryotic protein family. There are three TPPP paralogs in the human genome, denoted as TPPP1-TPPP3. TPPP1 and TPPP3 are intrinsically unstructured proteins (IUPs) that bind and polymerize tubulin and stabilize microtubules, but TPPP2 does not. Vertebrate...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.6b00902
更新日期:2017-02-21 00:00:00
abstract::NSC 613862 (S)-(-) and NSC 613863 (R)-(+) are the two chiral isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3, 4-b]pyrazin-7-yl carbamate. Both compounds bind to tubulin in a region that overlaps the colchicine site. They induce formation of abnormal polymers from purified GTP-Mg-tubulin, the active asse...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi970568t
更新日期:1998-01-13 00:00:00
abstract::The origin of the "S3" EPR signal from calcium-depleted photosystem 2 samples has been investigated. This signal is observed after freezing samples under illumination and has been assigned to an interaction between the manganese cluster and an oxidized histidine radical [Boussac et al. (1990) Nature 347; 303-306]. In ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00134a005
更新日期:1992-05-19 00:00:00
abstract::Binding of substrate to ornithine 4,5-aminomutase (OAM) and methylmalonyl-CoA mutase (MCM) leads to the formation of an electrostatic interaction between a conserved glutamate side chain and the adenosyl ribose of the adenosylcobalamin (AdoCbl) cofactor. The contribution of this residue (Glu338 in OAM from Clostridium...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi3012719
更新日期:2013-02-05 00:00:00