Abstract:
:It has been inferred from structural and computational studies that the mechanism of DNA polymerases involves subtle but important discrete steps that occur between binding and recognition of the correct dNTP and chemical catalysis. These steps potentially include local conformational changes involving active site residues, reorganization of Mg(2+)-coordinating ligands, and proton transfer. Here we address this broad issue by conducting extensive transient state kinetic analyses of DNA polymerase β (Pol β). We also performed kinetic simulations to evaluate alternative kinetic models. These studies provide some support for two-step subdomain closing and define constraints under which a kinetically significant prechemistry step can occur. To experimentally identify additional microscopic steps, we developed a stopped flow absorbance assay to measure proton formation that occurs during catalysis. These studies provide direct evidence that formation of the enzyme-bound 3'-O(-) nucleophile is rate determining for chemistry. We additionally show that at low pH the chemical step is rate limiting for catalysis, but at high pH, a postchemistry conformational step is rate limiting due to a pH-dependent increase in the rate of nucleotidyl transfer. Finally, we performed exhaustive analyses of [Mg(2+)] and pH effects. In contrast to published studies, the results suggest an irregular pH dependence of k(pol), which is consistent with general base catalysis involving cooperativity between two or more protonic residues. Overall, the results represent significant advancement in the kinetic mechanism of Pol β and also reconcile some computational and experimental findings.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Balbo PB,Wang EC,Tsai MDdoi
10.1021/bi200954rsubject
Has Abstractpub_date
2011-11-15 00:00:00pages
9865-75issue
45eissn
0006-2960issn
1520-4995journal_volume
50pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::Human apolipoprotein(a) kringle IV type 10 [apo(a) KIV(10)] contains a strong lysine-binding site (LBS) that mediates the interaction of Lp(a) with biological substrates such as fibrin. Mutations in the KIV(10) LBS have been reported in both the rhesus monkey and chimpanzee, and have been proposed to explain the lack ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi048156p
更新日期:2005-01-18 00:00:00
abstract::This paper reports on NMR-molecular mechanics structural studies of the (-)-trans-anti-[MC]dG adduct positioned opposite dC in the sequence context of the d(C1-C2-A3-T4-C5-[MC]G6-C7-T8-A9-C10-C11).d(G12-G13-T14++ +-A15-G16-C17-G18- A19-T20-G21-G22) duplex [designated (-)-trans-anti-[MC]dG.dC 11-mer duplex]. This adduc...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00018a029
更新日期:1995-05-09 00:00:00
abstract::The prokaryotic DNA protection during starvation (Dps) proteins typically protect macromolecules against damaging agents via physical association with DNA and by oxidizing and sequestering iron. However, Deinococcus radiodurans Dps-1, which binds DNA with high affinity, fails to protect DNA against hydroxyl radicals d...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi300703x
更新日期:2012-08-21 00:00:00
abstract::Campylobacter jejuni contains a general N-linked glycosylation pathway in which a heptasaccharide is sequentially assembled onto a polyisoprenyl diphosphate carrier and subsequently transferred to the asparagine side chain of an acceptor protein. The enzymes in the pathway function at a membrane interface and have in ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi701956x
更新日期:2007-12-18 00:00:00
abstract::Cyclopropane fatty acid (CFA) synthase of Escherichia coli catalyzes a modification of the acyl chains of phospholipid bilayers. We report (i) identification of the CFA synthase protein, (ii) overproduction (> 600-fold) and purification to essential homogeneity of the enzyme, and (iii) the amino acid sequence of CFA s...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00160a011
更新日期:1992-11-17 00:00:00
abstract::The classical complement pathway, which plays a vital role in preventing infection, is initiated by the action of the serine proteases C1r and C1s. We have examined the hydrolysis of substrates representing cleavage sequences in the physiological substrates for C1s, C2 and C4. These studies showed that the P(1)'-P(4)'...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi035507b
更新日期:2003-12-23 00:00:00
abstract::Myeloperoxidase (MPO) structural analysis has suggested that halides and pseudohalides bind to the distal binding site and serve as substrates or inhibitors, while others have concluded that there are two separate sites. Here, evidence for two distinct binding sites for halides comes from the bell-shaped effects obser...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0609725
更新日期:2007-01-16 00:00:00
abstract::Decorin-binding protein A (DBPA) is an important lipoprotein from the bacterium Borrelia burgdorferi, the causative agent of Lyme disease. The absence of DBPA drastically reduces the pathogenic potential of the bacterium, and biochemical evidence indicates DBPA's interactions with the glycosaminoglycan (GAG) portion o...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi3007093
更新日期:2012-10-23 00:00:00
abstract::Decapping is the first committed step in 5'-to-3' RNA decay, and in the cytoplasm of human cells, multiple decapping enzymes regulate the stabilities of distinct subsets of cellular transcripts. However, the complete set of RNAs regulated by any individual decapping enzyme remains incompletely mapped, and no consensus...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.0c00069
更新日期:2020-11-03 00:00:00
abstract::Increasing efforts are being invested in the construction of nanostructures with desired shapes and physical and chemical properties. Our strategy involves nanostructure design using naturally occurring protein building blocks. Inspection of the protein structural database (PDB) reveals the richness of the conformatio...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi061674a
更新日期:2007-02-06 00:00:00
abstract::We have studied the effect of melittin, a basic membrane-binding peptide, on Ca-ATPase activity and on protein and lipid dynamics in skeletal sarcoplasmic reticulum (SR), using time-resolved phosphorescence and fluorescence spectroscopy. Melittin completely inhibits Ca-ATPase activity, with half-maximal inhibition at ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00244a019
更新日期:1991-07-30 00:00:00
abstract::The electrons extracted from the CaMn(4) cluster during water oxidation in photosystem II are transferred to P(680)(+) via the redox-active tyrosine D1-Tyr161 (Y(Z)). Upon Y(Z) oxidation a proton moves in a hydrogen bond toward D1-His190 (His(Z)). The deprotonation and reprotonation mechanism of Y(Z)-OH/Y(Z)-O is of k...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi101364t
更新日期:2010-11-16 00:00:00
abstract::Human methionine synthase reductase (MSR), a diflavin oxidoreductase, plays a vital role in methionine and folate metabolism by sustaining methionine synthase (MS) activity. MSR catalyzes the oxidation of NADPH and shuttles electrons via its FAD and FMN cofactors to inactive MS-cob(II)alamin. A conserved aromatic resi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi2012228
更新日期:2011-12-27 00:00:00
abstract::Kinetic analysis and modeling studies of HIV-1 and HIV-2 proteinases were carried out using the oligopeptide substrate [formula: see text] and its analogs containing single amino acid substitutions in P3-P3' positions. The two proteinases acted similarly on the substrates except those having certain hydrophobic amino ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00135a008
更新日期:1992-05-26 00:00:00
abstract::The alpha, beta, and gamma subunits of the receptor with high affinity for immunoglobulin E were isolated and their compositions assessed by direct amino acid analysis and by incorporation of radioactive precursors. The compositions show no unusual features other than a rather high content of tryptophan in the alpha c...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00383a024
更新日期:1987-05-05 00:00:00
abstract::The structure of a 22 amino acid peptide, TPPI [Nedved, M. L., Gottlieb, P. A., & Moe, G. R. (1994) Nucleic Acids Res. 22, 5024-5030], that is similar to the proline repeat segment of the replication arrest protein, Tus, has been determined by 1H NMR in 50% trifluroethanol. The structure is a novel left-handed helix h...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi952419l
更新日期:1996-01-23 00:00:00
abstract::In this study, we have investigated the influence of regions outside the DNA-binding domain of the human glucocorticoid receptor on high-affinity DNA binding. We find that the DNA-binding domain shows a 10-fold lower affinity for a palindromic DNA-binding site than the intact receptor. The N-terminal part of the recep...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00152a047
更新日期:1992-09-22 00:00:00
abstract::Two nucleoside diphosphate analogs, 3'-C-methyl-ADP and 3'-C-methyl-UDP, have been tested as substrate and/or allosteric effectors using the adenosylcobalamin-dependent ribonucleoside diphosphate reductase of Corynebacterium nephridii. Neither analog was a substrate for the reductase. However, they did function as all...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00160a035
更新日期:1992-11-17 00:00:00
abstract::We have used enzymic and chemical probes to search for altered DNA conformations in the 5' flanking region of the gene for a high mobility group protein (HMG-T) from trout. This search was conducted in order to identify potential genetic elements that might be involved in the transcriptional control of the HMG-T gene....
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00402a012
更新日期:1988-01-26 00:00:00
abstract::To determine whether ligand-receptor interactions could engender the selective uptake by deficient cells of enzyme-laden liposomes, aggregated human IgG was used to coat liposomes which had previously trapped purified hexosaminidase A (Hex A). By a new, high-yield procedure, Hex A was purified 7000-fold from human pla...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00647a034
更新日期:1976-01-27 00:00:00
abstract::The kinetics of self-association for beef liver glutamate dehydrogenase (EC 1.4.1.3) have been measured by using pressure perturbation in both the time domain and the frequency domain by monitoring scattered light intensity. The kinetic behavior is entirely consistent with the random self-association model proposed by...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00579a007
更新日期:1979-06-12 00:00:00
abstract::The spin-1/2-carrying NO molecule interacts with both the S1 and S2 states of the water oxidizing complex. The intermediates of the interaction can be resolved and trapped by NO treatment at subzero temperatures. At -30 degrees C and in the presence of approx. 500-700 microM NO, S1 loses the ability to yield by illumi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi962899w
更新日期:1997-07-29 00:00:00
abstract::Big ET-1 and big ET-3 are precursor peptides which render endothelin-1 (ET-1) and endothelin-3 (ET-3) relatively unreactive and resistant to proteolytic cleavage. Big ET-1 is cleaved in vivo by ECE-1 (endothelin-converting enzyme), and big ET-3 is also cleaved but apparently to a significantly lesser extent by this en...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi981689b
更新日期:1999-02-09 00:00:00
abstract::Histone H10 differs substantially from H1 in a 37-residue region of the primary structure that is conserved between the usual H1 histones of fish and mammals. Instead, H10 is homologous to H5 in this region. Histones H10 and H5 might be classified together in a histone category distinct from the usual H1. ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00511a035
更新日期:1981-04-14 00:00:00
abstract::The properties of the Fe and Mo sites of the iron-molybdenum cofactor of nitrogenase with respect to binding and activation of N(2) have been studied by molecular mechanics calculations on the local protein environment and by density functional theory (DFT) calculations on subsections of the cofactor. The DFT calculat...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi025623z
更新日期:2002-11-26 00:00:00
abstract::A detailed study has been made of the polyacrylamide gel electrophoresis of DNA restriction fragments obtained from two plasmids, pBR322 and p82-6B. Variables studied were molecular weight, gel concentration, temperature, and electric field strength. The retardation coefficients of the larger fragments (greater than 8...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00295a023
更新日期:1983-12-20 00:00:00
abstract::ATP hydrolysis by the maltose transporter (MalFGK(2)) is regulated by maltose binding protein (MBP). Binding of maltose to MBP brings about a conformational change from open to closed that leads to a strong stimulation of the MalFGK(2) ATPase. In this study, we address the long-standing but enigmatic observation that ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9007066
更新日期:2009-08-25 00:00:00
abstract::The existence of bacterial K(+)/H(+) antiporters that prevent the overaccumulation of potassium in the cytoplasm was predicted by Peter Mitchell almost 50 years ago. The importance of K(+)/H(+) antiport for bacterial physiology is widely recognized, but its molecular mechanisms remain underinvestigated. Here, we demon...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi902173y
更新日期:2010-03-23 00:00:00
abstract::Tropomyosin is postulated to confer stability to actin filaments in nonmuscle cells. We have found that a nonmuscle tropomyosin isolated from the intestinal epithelium can directly stabilize actin filaments by slowing depolymerization from the pointed, or slow-growing, filament end. Kinetics of elongation and depolyme...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00447a035
更新日期:1989-10-17 00:00:00
abstract::The binding of various forms of vanadate to myosin and myosin subfragment 1 (S-1) was studied by 51V NMR at increasing vanadate concentrations between 0.06 and 1.0 mM. The distribution of the various forms of vanadate in the solution depended on the total concentration of vanadate. At low concentrations, the predomina...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00490a029
更新日期:1990-09-25 00:00:00