Telomerase is a ribonucleoprotein enzyme complex that adds 5'-TTAGGG-3' repeats onto the ends of human chromosomes, providing a telomere maintenance mechanism for approximately 90% of human cancers. We have purified human telomerase approximately 10(8)-fold, with the final elution dependent on the enzyme's ability to catalyze nucleotide addition onto a DNA oligonucleotide of telomeric sequence, thereby providing specificity for catalytically active telomerase. Mass spectrometric sequencing of the protein components and molecular size determination indicated an enzyme composition of two molecules each of telomerase reverse transcriptase, telomerase RNA, and dyskerin.
catalytically
R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA-DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA-DNA hybrids. GFP-dRNH1 binds strongly to RNA-DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA-DNA hybrids under a wide range of conditions.
KatG dismutates hydrogen peroxide in a nonscrambling mechanism such that both oxygen atoms of dioxygen derive from the same molecule of hydrogen peroxide (16, 17). Unlike classical catalases, however, which do not accumulate intermediates during H2O2 turnover because of very rapid rates of both the peroxide reduction and the peroxide oxidation steps, KatG forms a species characteristic of oxyferrous heme (peroxidase Cmpd III) in the presence of high concentrations of peroxide (11, 15, 18). This intermediate is usually stable in peroxidases and is considered to be catalytically inert, but it must be highly unstable in KatG because catalase turnover occurs while the heme is in this form (In cytochrome c peroxidase oxyferrous heme is unstable because of internal redox chemistry involving Trp191, but cytochrome c peroxidase does not exhibit catalase activity (19).) and the catalase activity of the W321F mutant of M. tuberculosis KatG is only moderately reduced (20). In the companion paper (69), formation of oxyenzyme from the carbonyl enzyme shows that in WT KatG, oxyferrous heme is too stable to be a viable catalase intermediate unless some other unique feature produced in the oxyferrous enzyme formed in the presence of hydrogen peroxide alters its properties. A protein-based radical proposed here to be formed on the distal side amino acid adduct, which co-exists with oxyferrous heme when peroxide turnover occurs, is suggested to be a catalytically competent intermediate in catalase turnover in WT KatG.
Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30, providing the structural distortion needed for R-loop formation.
The catalytically inactivating subset within rabbit serum polyclonal antibody to the solubilized, purified 55,000 to 60,000 dalton active fragment of rat liver microsomal β-hydroxy-β-methylglutaryl coenzyme A reductase immunoinactivates this enzyme with little or no diminution of effect by enzyme catalytically inactivated by incubation of microsomes with ATP,Mg++. Reactivation of inactive enzyme with ethanol-treated rat liver phosphatase restores antibody affinity showing that the catalytically inactivating subset of antibody exhibits marked or complete affinity for the active enzyme over the ATP,Mg++-inactivated form. This means that immunoinactivation using this antibody is not a valid way of measuring changes in the specific activity of the enzyme via phosphorylation-dephosphorylation. Preference for the active enzyme has not been obvious because when different amounts of enzyme activity are used in immunotitrations of samples of low activity, apparent differences in specific activity are observed when none actually exist. If precautions are not taken, results are obtained supporting phosphorylation by using an antibody that is not capable of distinguishing it.
Escherichia coli DNA photolyase catalyzes the light-driven (300-500 nm) repair of pyrimidine dimers formed between adjacent pyrimidine bases in DNA exposed to UV light (200-300 nm). The light-driven repair process is facilitated by two enzyme-bound cofactors, FADH2 and 5,10-methenyltetrahydrofolate. The function of the folate has been characterized in greater detail in this series of experiments. Investigations of the relative binding affinities of photolyase for the monoglutamate and polyglutamate forms of 5,10-methenyltetrahydrofolate show that the enzyme has a greater affinity for the naturally occurring polyglutamate forms of the folate and that the exogenously added monoglutamate derivative is less tightly associated with the protein. Multiple turnover experiments reveal that the folate remains bound to photolyase even after 10 turnovers of the enzyme. Examination of the rates of repair by photolyase containing stoichiometric folate in the presence or absence of free folate under multiple turnover conditions and at micromolar concentrations of enzyme also demonstrates that the folate acts catalytically. The stimulation of turnover by exogenous folate seen at low concentrations of photolyase is shown to be due to the lower affinity of photolyase for the monoglutamate derivative used in reconstitution procedures. These results demonstrate that the folate of E. coli DNA photolyase is a bona fide cofactor and does not decompose or dissociate during multiple turnovers of the enzyme.
A heterogeneous catalyst has active sites, which are the atoms or crystal faces where the reaction actually occurs. Depending on the mechanism, the active site may be either a planar exposed metal surface, a crystal edge with imperfect metal valence, or a complicated combination of the two. Thus, not only most of the volume but also most of the surface of a heterogeneous catalyst may be catalytically inactive. Finding out the nature of the active site requires technically challenging research. Thus, empirical research for finding out new metal combinations for catalysis continues. 2ff7e9595c
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