Abstract:
:Conformational dynamics are thought to be a prerequisite for the catalytic activity of enzymes. However, the exact relationship between structural fluctuations and function is not well understood. In this work hydrogen/deuterium exchange (HDX) and electrospray ionization mass spectrometry (ESI-MS) are used for exploring the conformational dynamics of thermolysin. Amide HDX reflects the internal mobility of proteins; regions that undergo frequent unfolding-refolding show faster exchange than segments that are highly stable. Thermolysin is a zinc protease with an active site that is located between two lobes. Substrate turnover is associated with hinge bending that leads to a closed conformation. Product release regenerates the open form, such that steady-state catalysis involves a continuous closing/opening cycle. HDX/ESI-MS with proteolytic peptide mapping in the absence of substrate shows that elements in the periphery of the two lobes are most mobile. A comparison with previous X-ray data suggests that these peripheral regions undergo quite pronounced structural changes during the catalytic cycle. In contrast, active site residues exhibit only a moderate degree of backbone flexibility, and the central zinc appears to be in a fairly rigid environment. The presence of both rigid and moderately flexible elements in the active site may reflect a carefully tuned balance that is required for function. Interestingly, the HDX behavior of catalytically active thermolysin is indistinguishable from that of the free enzyme. This result is consistent with the view that catalytically relevant motions preexist in the resting state and that enzyme function can only be performed within the limitations given by the intrinsic dynamics of the protein. The data presented in this work indicate the prevalence of stochastic elements in the function of thermolysin, rather than supporting a deterministic mechanism.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Liu YH,Konermann Ldoi
10.1021/bi800463qsubject
Has Abstractpub_date
2008-06-17 00:00:00pages
6342-51issue
24eissn
0006-2960issn
1520-4995journal_volume
47pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::PII proteins are sensors of alpha-ketoglutarate and adenylylate energy charge that regulate signal transduction proteins, metabolic enzymes, and permeases involved in nitrogen assimilation. Here, purified Escherichia coli PII and two of its receptors, ATase and NRII, were used to study the mechanisms of sensation by P...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9011594
更新日期:2009-12-08 00:00:00
abstract::The interaction of myristoyllysophosphatidylcholine with bovine myelin basic protein at pH 7.4 and 4.5, I = 0.48, has been investigated by a recycling partition equilibrium technique with Bio-Gel P-2 as the gel phase. Important points to emerge from this direct binding study are that it is a monomeric (not micellar) a...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00378a002
更新日期:1987-02-24 00:00:00
abstract::The serotonin transporter (SERT) is a member of a highly homologous family of proteins responsible for the reuptake of biogenic amines from the synaptic cleft. We took advantage of native restriction sites in SERT to construct a chimeric transporter containing a small (34 amino acid) region of the norepinephrine trans...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi962150l
更新日期:1997-02-11 00:00:00
abstract::In a large number of plant species hydroxynitrile lyases catalyze the decomposition of cyanohydrins in order to generate hydrogen cyanide upon tissue damage. Hydrogen cyanide serves as a deterrent against herbivores and fungi. In vitro hydroxynitrile lyases are proficient biocatalysts for the stereospecific synthesis ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi802162s
更新日期:2009-04-21 00:00:00
abstract::The prion protein (PrP) is a cell-surface Cu2+ binding glycoprotein which can bind six copper ions. The role of Cu2+ in PrP function, misfolding, and prion disease has generated much interest; however, the field has been hampered by a lack of consensus with regard to the affinity of Cu2+ for PrPC. Here we build on our...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9011397
更新日期:2009-09-29 00:00:00
abstract::Polyhistidine affinity tags are routinely employed as a convenient means of purifying recombinantly expressed proteins. A tacit assumption is commonly made that His tags have little influence on protein structure and function. Attachment of a His tag to the N-terminus of the robust globular protein myoglobin leads to ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi2003923
更新日期:2011-06-28 00:00:00
abstract::The effects of cysteine modification and variations in pH on the equilibrium parameters for inducer and operator binding to the lactose repressor protein were examined. Operator binding affinity was minimally affected by increasing the pH from 7.5 to 9.2, whereas inducer binding was decreased for both the unliganded p...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00367a020
更新日期:1986-09-23 00:00:00
abstract::The newly discovered oncogenic protein gankyrin, which contains six ankyrin repeats, has been reported to be involved in the phosphorylation and degradation of the retinoblastoma gene product, Rb. Using in vitro systems, we have identified a peptide fragment of gankyrin, 176LHLACDEERN185, which is responsible for bind...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi011550s
更新日期:2002-03-26 00:00:00
abstract::Pump-1 cDNA has recently been isolated by screening a human tumor cDNA library with a transin (rat stromelysin) probe under low-stringency hybridization conditions. The cDNA codes for a potential protein with significant sequence similarity to the metalloproteinases collagenase and stromelysin, but which lacks the hem...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00439a004
更新日期:1989-06-27 00:00:00
abstract::B₁₂-dependent enzymes employ radical species with exceptional prowess to catalyze some of the most chemically challenging, thermodynamically unfavorable reactions. However, dealing with highly reactive intermediates is an extremely demanding task, requiring sophisticated control strategies to prevent unwanted side rea...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi500050k
更新日期:2014-06-17 00:00:00
abstract::The phosphorelay signal transduction pathway controls sporulation initiation in Bacillus subtilis. Transfer of a phosphoryl group from multiple kinases (KinA and KinB) through a single domain response regulator homologue (Spo0F), a phosphotransferase (Spo0B), and ultimately to a transcriptional regulator, (Spo0A) acti...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi981340o
更新日期:1998-11-24 00:00:00
abstract::DNA gyrase and topoisomerase IV (Topo IV) are cellular targets of quinolone antibacterial drugs. The Ser-80 and the Glu-84 of the ParC subunit have been identified as mutational hotspots for quinolone resistance. Mutant Topo IV proteins containing a quinolone resistance-conferring mutation have been constructed, and t...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi026352v
更新日期:2002-10-01 00:00:00
abstract::Solvent kinetic isotope effect studies of electron transfer within xanthine oxidase have been performed, using a stopped-flow pH-jump technique to perturb the distribution of reducing equivalents within partially reduced enzyme and follow the kinetics of reequilibration spectrophotometrically. It is found that the rat...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00099a004
更新日期:1991-09-03 00:00:00
abstract::Syk, a nonreceptor protein-tyrosine kinase, is activated by both oxidative and osmotic stress and plays different roles in the transduction of stress signals. In this study, the regulation of oxidative and osmotic stress induced Syk activation was investigated utilizing Syk-negative DT40 cells, expressing various Syk ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9729460
更新日期:1998-04-21 00:00:00
abstract::A new method of calculation, based on a direct fitting of the protein fluorescence intensity observed upon coenzyme binding (H.-P. Lutz, unpublished results), is used to study the negative cooperative behavior of glyceraldehyde-3-phosphate dehydrogenase from sturgeon muscle. The calculation procedure simultaneously el...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00288a014
更新日期:1983-09-13 00:00:00
abstract::The amyloid beta-protein (1-42) is a major constituent of the abnormal extracellular amyloid plaque that characterizes the brains of victims of Alzheimer's disease. Two peptides, with sequences derived from the previously unexplored C-terminal region of the beta-protein, beta 26-33 (H2N-SNKGAIIG-CO2H) and beta 34-42 (...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00463a003
更新日期:1990-03-20 00:00:00
abstract::In the ATP-dependent carboxylation of biotin that is catalyzed by most biotin-dependent carboxylases, a fundamental mechanistic question is whether the ATP activates bicarbonate (via the formation of carboxyphosphate as an intermediate) or whether the ATP activates biotin (via the formation of O-phosphobiotin). We hav...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00421a009
更新日期:1988-10-18 00:00:00
abstract::Gemcitabine 5'-diphosphate (F(2)CDP) is a potent inhibitor of ribonucleotide reductases (RNRs), enzymes that convert nucleotides (NDPs) to deoxynucleotides and are essential for DNA replication and repair. The Escherichia coli RNR, an alpha2beta2 complex, when incubated with 1 equiv of F(2)CDP catalyzes the release of...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi901590q
更新日期:2009-12-15 00:00:00
abstract::The specific rate of superoxide (O2(•-)) production in the purified active crystallizable cytochrome b6f complex, normalized to the rate of electron transport, has been found to be more than an order of magnitude greater than that measured in isolated yeast respiratory bc1 complex. The biochemical and structural basis...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi4013534
更新日期:2013-12-17 00:00:00
abstract::Nuclear magnetic resonance (NMR) (15)N relaxation methods have been used to characterize the backbone dynamics of the N-terminal core domain of the HIV-1 capsid protein (CA(151)). The domain, which has an unusually flat, triangular shape, tumbles in solution at 28 degrees C with an effective rotational correlation tim...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi990991x
更新日期:1999-08-10 00:00:00
abstract::Comparisons of membrane glycopeptides from baby hamster kidney fibroblasts (BHK21/C13) and a clone transformed by Rous sarcoma virus (C13/B4) were made by using cells metabolically labeled with radioactive D-glucose and L-fucose. Most of the glycopeptides were metabolically labeled with both the general and the specif...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00579a015
更新日期:1979-06-12 00:00:00
abstract::The self-association of rabbit muscle phosphofructokinase at pH 7.0 was investigated by velocity sedimentation. The process was demonstrated to be in a rapid, dynamic equilibrium. The concentration dependence of the weight-average sedimentation coefficient was monitored within the range of 10-750 microgram/mL. The sed...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00513a040
更新日期:1981-05-12 00:00:00
abstract::NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of RK-1, an antimicrobial peptide from rabbit kidney recently discovered from homology screening based on the distinctive physicochemical properties of the corticostatins/defensins. RK-1 consists of 32 res...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi000457l
更新日期:2000-12-26 00:00:00
abstract::G-Rich DNA and RNA have a higher propensity to form G-quadruplex structures, but the presence of G-runs alone is not sufficient to prove that such sequences can form stable G-quadruplexes. While G-rich sequences are essential for G-quadruplex formation, not all G-rich sequences have the propensity to form G-quadruplex...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi500773c
更新日期:2014-11-18 00:00:00
abstract::Intrinsic factor (IF(50)) is a cobalamin (Cbl)-transporting protein of 50 kDa, which can be cleaved into two fragments: the 30 kDa N-terminal peptide IF(30) and the 20 kDa C-terminal glycopeptide IF(20). Experiments on binding of Cbl to IF(30), IF(20), and IF(50) revealed comparable association rate constants (k(+)(Cb...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi047936v
更新日期:2005-03-08 00:00:00
abstract::We reacted uracil-containing M13mp2 DNA with N-hydroxy-2-aminofluorene to produce a template with N-(deoxyguanosin-8-yl)-2-aminofluorene adducts. This template was hybridized to a non-uracil-containing linear fragment from which the lac z complementing insert had been removed to produce a gapped substrate. DNA synthes...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00433a014
更新日期:1989-04-04 00:00:00
abstract::We have used 2D 1H NMR to determine the structure of glucagon-like peptide-1-(7-36) amide bound to a dodecylphosphocholine micelle. In this membranelike environment, the peptide hormone is shown to have a structure similar to that observed for glucagon. It consists of an N-terminal random coil segment (residues 1-7), ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00178a009
更新日期:1994-03-29 00:00:00
abstract::Subunit II of the aa3 type cytochrome c oxidase contains a binuclear copper center (CuA) which functions as the entry point for electrons donated by cytochrome c. We have introduced site-specific mutations in residues liganding the CuA center in the oxidase of the bacterium Paracoccus denitrificans; the purified, full...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi962040e
更新日期:1997-03-18 00:00:00
abstract::The sterol (free and esterified cholesterol) transport activity of the small-intestinal brush border membrane was solubilized with the short-chain detergent diheptanoylphosphatidylcholine and reconstituted to an artificial membrane system (proteoliposomes). The resulting proteoliposomes were identified as unilamellar ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi970625i
更新日期:1997-09-02 00:00:00
abstract::Sarcosine oxidase from Corynebacterium sp. P-1 binds 2 mol of tetrahydrofolate/mol of enzyme (KD = 8.8 microM). The same stoichiometry is observed with tetrahydropteroyltetraglutamate (KD = 15.4 microM). Binding is also observed with pteroyltetraglutamate and with 5-formyltetrahydrofolate. In the case of the pteroylmo...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00397a029
更新日期:1987-11-17 00:00:00