Abstract:
:The catalytically competent active-site structure of a true acylenzyme reaction intermediate of TEM-1 beta-lactamase formed with the kinetically specific spin-labeled substrate 6-N-(2,2,5,5-tetramethyl-1-oxypyrrolinyl-3-carboxyl)-penicillanic acid isolated under cryoenzymologic conditions has been determined by angle-selected electron nuclear double resonance (ENDOR) spectroscopy. Cryoenzymologic experiments with use of the chromophoric substrate 6-N-[3-(2-furanyl)-propen-2-oyl]-penicillanic acid showed that the acylenzyme reaction intermediate could be stabilized in the -35 to -75 degrees C range with a half-life suitably long to allow freeze-quenching of the reaction species for ENDOR studies while a noncovalent Michaelis complex could be optically identified at temperatures only below -70 degrees C. The wild-type, Glu166Asn, Glu240Cys, and Met272Cys mutant forms of the mature enzyme were overexpressed in perdeuterated minimal medium to allow detection and assignment of proton resonances specific for the substrate and chemically modified amino acid residues in the active site. From analysis of the dependence of the ENDOR spectra on the setting of the static laboratory magnetic field H0, the dipolar contributions to the principal hyperfine coupling components were estimated to calculate the separations between the unpaired electron of the nitroxyl group and isotopically identified nuclei. These electron-nucleus distances were applied as constraints to assign the conformation of the substrate in the active site and of amino acid side chains by molecular modeling. Of special interest was that the ENDOR spectra revealed a water molecule sequestered in the active site of the acylenzyme of the wild-type protein that was not detected in the deacylation impaired Glu166Asn mutant. On the basis of the X-ray structure of the enzyme, the ENDOR distance constraints placed this water molecule within hydrogen-bonding distance to the carboxylate side chain of glutamate-166 as if it were poised for nucleophilic attack of the scissile ester bond. The ENDOR results provide experimental evidence of glutamate-166 in its functional role as the general base catalyst in the wild-type enzyme for hydrolytic breakdown of the acylenzyme reaction intermediate of TEM-1 beta-lactamase.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Mustafi D,Sosa-Peinado A,Makinen MWdoi
10.1021/bi0021075subject
Has Abstractpub_date
2001-02-27 00:00:00pages
2397-409issue
8eissn
0006-2960issn
1520-4995pii
bi0021075journal_volume
40pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::In this paper the interaction of cytoplasmic CopZ and the N-terminal domain of the CopA ATPase from Bacillus subtilis has been studied by NMR through (15)N-(1)H HSQC experiments in order to understand the role of the two proteins in the whole copper trafficking mechanism of the bacteria. It appears that the two protei...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi027096p
更新日期:2003-02-25 00:00:00
abstract::Six rhodopsin mutants containing disulfide cross-links between different cytoplasmic regions were prepared: disulfide bond 1, between Cys65 (interhelical loop I-II) and Cys316 (end of helix VII); disulfide bond 2, between Cys246 (end of helix VI) and Cys312 (end of helix VII); disulfide bond 3, between Cys139 (end of ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9912443
更新日期:1999-09-28 00:00:00
abstract::Ketosteroid isomerase (KSI) is one of the most proficient enzymes catalyzing an allylic isomerization reaction at a diffusion-controlled rate. In this study of KSI, we have detailed the structures of its active site, the role of various catalytic residues, and have explained the origin of the its fast reactivity by ca...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi001629h
更新日期:2000-11-14 00:00:00
abstract::The envelope glycoprotein of HIV-1 consists of the surface subunit gp120 and the transmembrane subunit gp41. Binding of gp120 to target cell receptors induces a conformational change in gp41, which then mediates the fusion of viral and cellular membranes. A buried isoleucine (Ile573) in a central trimeric coiled coil ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0024759
更新日期:2001-03-06 00:00:00
abstract::A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calm...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00318a018
更新日期:1984-11-06 00:00:00
abstract::Quinone oxidoreductase 2 (QR2) purified from human red blood cells was recently shown to be a potential target of the quinoline antimalarial compounds [Graves et al., (2002) Mol. Pharmacol. 62, 1364]. QR2 catalyzes the two-electron reduction of menadione via the oxidation of N-alkylated or N-ribosylated nicotinamides....
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi035923w
更新日期:2004-04-20 00:00:00
abstract::In the DNA recognition mode of C(2)H(2)-type zinc fingers, the finger-finger connection region, consisting of the histidine spacing (HX(3-5)H) and linker, would be important for determining the orientation of the zinc finger domains. To clarify the influence of spacing between two ligand histidines in the DNA binding,...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi035999s
更新日期:2004-05-25 00:00:00
abstract::The electric organ of the ray, Torpedo ocellata, can serve as a source for both the acetylcholine (ACh) receptor and its ionic channel. The two entities were identified by their specific binding of [3H]ACh and [3H]perhydrohistrionicotoxin ([3H]H12-HTX), respectively. Binding of [3H]H12-HTX was inhibited by certain dru...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00618a023
更新日期:1978-12-12 00:00:00
abstract::The heterotrimeric G protein subunit, alpha(s), can move reversibly from plasma membranes to cytoplasm in response to activation by GPCRs or activating mutations. We examined the importance of the unique N-terminus of alpha(s) in this translocation in cultured cells. alpha(s) contains a single site for palmitoylation ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi025533u
更新日期:2002-07-30 00:00:00
abstract::Bacteriophage T7 codes for a single-stranded DNA binding protein. This protein is the product of gene 2.5 and has been found previously to stimulate specifically the activity of the phage-coded DNA polymerase. We report here that the T7 DNA binding protein also stimulates the activity of the phage-coded exonuclease. T...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00266a042
更新日期:1982-11-09 00:00:00
abstract::The amino acid sequence of the peptide alpha1(I)-CB6B, the carboxyl-terminal peptide containing 91 residues of alpha1(I) chain of chick skin collagen, is described. The amino acid sequence was determined by automated Edman degradation of intact peptide plus its tryptic and thermolytic peptides. The comparison of this ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00619a018
更新日期:1978-12-26 00:00:00
abstract::The membrane-active, cationic, β-hairpin peptide, arenicin, isolated from marine polychaeta Arenicola marina exhibits a broad spectrum of antimicrobial activity. The peptide in aqueous solution adopts the significantly twisted β-hairpin conformation without pronounced amphipathicity. To assess the mechanism of arenici...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi200746t
更新日期:2011-07-19 00:00:00
abstract::Properties of the interaction of bleomycin (Blm) and metallobleomycins [M = Zn, Cu(II), Fe(III), and HO(2)-Co(III)] with site-specific and nonspecific DNA oligomers, d(GGAAGCTTCC)(2) (I) and d(GGAAATTTCC)(2) (II), respectively, were investigated. With both 10-mers association constants increased in the series Blm A(2)...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi001915t
更新日期:2001-06-26 00:00:00
abstract::Acid beta-glucosidase is a lysosomal membrane protein that cleaves the O-beta-D-glucosidic linkage of glucosylceramide and aryl-beta-glucosides. Full activity reconstitution of the pure enzyme requires phospholipids and saposin C, an 80 aa activator protein. The deficiency of the enzyme or activator leads to Gaucher d...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi980785+
更新日期:1998-08-18 00:00:00
abstract::We have developed a model of glycophorin in a phospholipid bilayer membrane in order to study the thermodynamics of this system and to understand the detailed behavior of recent calorimetric data. We assume that the larger glycophorin polar group can be considered as either adopting a pancakelike conformation at the b...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00381a019
更新日期:1987-04-07 00:00:00
abstract::The interaction of cholesterol with phospholipids has been studied with a variety of techniques; however, the possible consequences of such interactions in vivo have not been demonstrated. In this study, the cholesterol-dependent absorbance spectrum of cytochrome P-450scc was used to monitor cholesterol availability i...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00363a017
更新日期:1986-07-29 00:00:00
abstract::The interactions of the ligand binding domain (LBD) of androgen receptor (AR) and the AR T877A mutant, found in prostate cancer, with peptides from coactivator and corepressor proteins or random phage display peptides were investigated using in vitro time-resolved fluorescence resonance energy transfer (TR-FRET). Inte...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi061321b
更新日期:2007-01-23 00:00:00
abstract::We report here that polyamide nucleic acid (PNA) as well as a polyamide nucleic acid-DNA chimera complementary to the primer binding site of the HIV-1 genome can completely block priming by tRNA3Lys and consequently the in vitro initiation of reverse transcription by HIV-1 RT. Conventional heating and cooling is not r...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi972197m
更新日期:1998-01-20 00:00:00
abstract::Many current models of the Q cycle for the cytochrome (cyt) b6f and the cyt bc1 complexes incorporate 'Rieske' iron-sulfur protein (ISP) domain movements to gate electron transfer and to ensure high yields of proton shuttling. It was previously proposed that copper ions, which bind at a site distant from the quinol ox...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi015996k
更新日期:2002-03-26 00:00:00
abstract::Complete 1H NMR sequential assignments have been made for copper(I)- and silver (I)-substituted metallothionein (MT) from Saccharomyces cerevisiae using standard 2D 1H NMR methods. The fingerprint region of the COSY spectrum of both metalloproteins shows a doubling of a few backbone proton resonances from residue K41 ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00077a032
更新日期:1993-07-06 00:00:00
abstract::The folding and unfolding rates of the small protein, barstar, have been monitored using stopped-flow measurements of intrinsic tryptophan fluorescence at 25 degrees C, pH 8.5, and have been compared over a wide range of urea and guanidine hydrochloride (GdnHCl) concentrations. When the logarithms of the rates of fold...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi011494v
更新日期:2002-02-05 00:00:00
abstract::The ion binding parameters of the EF-hand Ca2+ binding motif are carefully tuned for different biological applications. The present study examines the contribution of the ninth position of the Ca2+-coordinating EF-loop to the tuning of Ca2+ affinity and selectivity, using the model EF-loop of the Escherichia coli gala...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi952430l
更新日期:1996-05-28 00:00:00
abstract::The potential of the frequently encountered (βα)8-barrel fold to acquire new functions was tested by an approach combining random mutagenesis and selection in vivo. For this purpose, the genes encoding 52 different phosphate-binding (βα)8-barrel proteins were subjected to error-prone PCR and cloned into an expression ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.9b00579
更新日期:2019-10-15 00:00:00
abstract::We have studied the pathway and kinetics of refolding of recombinant human brain-derived neurotrophic factor (BDNF), which is a very tightly-associated dimer in its native state. When BDNF unfolded in 6 M guanidine hydrochloride is diluted 20-fold into phosphate-buffered saline, a partially folded intermediate is rapi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00091a036
更新日期:1993-10-12 00:00:00
abstract::Cytochrome b(561) in adrenal chromaffin vesicle membranes conveys electron equivalents from extravesicular ascorbate to the intravesicular monodehydroascorbate radical. We conducted a stopped-flow study on the reaction of ascorbate with purified cytochrome b(561) in the detergent-solubilized state for the first time. ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0267588
更新日期:2003-07-15 00:00:00
abstract::Phospholipase D has been purified 680-fold from an acetone powder of savoy cabbage in an overall yield of 30%. The purification involves solubilization of the acetone powder in a Ca2+-containing buffer and subsequent ammonium sulfate fractionation. Gel filtration on Sephadex G-200 and hydrophobic affinity chromatograp...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00591a014
更新日期:1979-11-27 00:00:00
abstract::Aerobic and facultative bacteria and archaea harboring mer loci exhibit resistance to the toxic effects of Hg(II) and organomercurials [RHg(I)]. In broad spectrum resistance, RHg(I) is converted to less toxic Hg(0) in the cytosol by the sequential action of organomercurial lyase (MerB: RHg(I) → RH + Hg(II)) and mercur...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi100802k
更新日期:2010-09-21 00:00:00
abstract::Homology modeling was used to identify two particular residues, Glu175 and Ala176, in Pseudomonas stutzeri phosphite dehydrogenase (PTDH) as the principal determinants of nicotinamide cofactor (NAD(+) and NADP(+)) specificity. Replacement of these two residues by site-directed mutagenesis with Ala175 and Arg176 both s...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi035018b
更新日期:2003-10-14 00:00:00
abstract::Oligodeoxynucleotides modified site-specifically with dG-(+)-trans- and dG-(+)-cis-anti-BPDE (7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) or dG-(-)-trans- and dG-(-)-cis-anti-BPDE were used as templates in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I....
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00080a027
更新日期:1993-07-27 00:00:00
abstract::The enzymes in the catechol meta-fission pathway have been studied for more than 50 years in several species of bacteria capable of degrading a number of aromatic compounds. In a related pathway, naphthalene, a toxic polycyclic aromatic hydrocarbon, is fully degraded to intermediates of the tricarboxylic acid cycle by...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.6b00050
更新日期:2016-05-10 00:00:00