Abstract:
:The reversible inhibition of beta-lactamase I from Bacillus cereus by cloxacillin, methicillin, and nafcillin has been systematically investigated. For these substrates the enzymatic reaction involves partitioning of the substrate between turnover and inhibition. Typically, concentrations of several hundred millimolar are necessary for complete inactivation. The completely inactivated enzyme could be formed by incubation at temperatures above 20 degrees C, where inhibition competes more effectively with turnover, and then stabilized by dropping the temperature to 0 degrees C or lower. The inactivated enzyme was rapidly separated from unreacted substrate and product at low temperature by centrifugal gel filtration or ion exchange and examined by far-UV circular dichroism for evidence of a conformational change. At pH 7 the inactivated enzyme had a secondary structure essentially identical with that of the native enzyme. The fluorescence emission spectrum of the inactivated enzyme (at pH 7) was also identical with that of the native enzyme. However, the inactivated enzyme was found to be considerably more sensitive to thermal denaturation, to acid-induced conformational isomerization, and to trypsinolysis than the native enzyme. We conclude from the circular dichroism results that the structure of the reversibly inactivated enzyme cannot be significantly different from that of the native enzyme. Therefore, previous findings that have been interpreted as indicating a major conformational change must be reevaluated. From examination of the low-resolution crystallographic structure of the enzyme we propose that the most likely cause of the inactivation is an alternate conformational state of the acyl-enzyme intermediate involving movement of one or more of the alpha-helices forming part of the active site. Such a structural effect could leave the secondary structure unchanged but have significant effects on the tertiary structure, catalysis, mobility, and susceptibility to trypsin and denaturation. We propose that the underlying physical reason why certain beta-lactam substrates bring about this "substrate-induced deactivation", or suicide inactivation, of the enzyme is due to the presence of the alternative acyl-enzyme conformation of similar free energy to the productive one, in which one (or more) essential catalytic group is no longer optimally oriented for catalyzing deacylation. Thus for substrates with a slow rate of deacylation (less than or equal to 100 s-1) the conformational transition can compete effectively on the time scale of the turnover reaction.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Fink AL,Behner KM,Tan AKdoi
10.1021/bi00388a011subject
Has Abstractpub_date
1987-07-14 00:00:00pages
4248-58issue
14eissn
0006-2960issn
1520-4995journal_volume
26pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::A simple and rapid procedure is described for purification of P-glycoprotein (Pgp) from a multidrug-resistant Chinese hamster ovary cell line (CR1R12) in which the plasma membranes are highly enriched in Pgp (Al-Shawi, M.K., Senior A.E. (1993) J. Biol. Chem, 268, 4197-4206). The procedure consisted of octylglucoside s...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00189a008
更新日期:1994-06-14 00:00:00
abstract::Escherichia coli PurK, a dimeric N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) synthetase, catalyzes the conversion of 5-aminoimidazole ribonucleotide (AIR), ATP, and bicarbonate to N5-CAIR, ADP, and Pi. Crystallization of both a sulfate-liganded and the MgADP-liganded E. coli PurK has resulted in structures at 2....
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi991618s
更新日期:1999-11-23 00:00:00
abstract::Hydrogen exchange of the individual amide protons of alanine-90 (F5), glutamine-91 (F6), serine-92 (F7), and histidine-93 (F8) residues in cyanometmyoglobin of sperm whale has been studied by 1H nuclear magnetic resonance spectroscopy at 360 MHz. The amide proton resonance of F5, F6, and F7 have been assigned by use o...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00347a024
更新日期:1985-12-17 00:00:00
abstract::Pokeweed antiviral protein (PAP) is a single-chain ribosome inactivating protein (RIP) that binds to ribosomes and depurinates the highly conserved alpha-sarcin/ricin loop (SRL) of the large subunit rRNA. Catalytic depurination of a specific adenine has been proposed to result in translation arrest and cytotoxicity. H...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi048188c
更新日期:2005-02-22 00:00:00
abstract::The objective of this work is to determine the molecular mechanism and regulation of short-chain acyl-CoA dehydrogenase (SCAD) from Megasphaera elsdenii. To achieve this, the gene coding for SCAD from M. elsdenii was cloned and sequenced. Site-directed mutagenesis was then used to identify an amino acid residue that i...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00091a026
更新日期:1993-10-12 00:00:00
abstract::The solution conformation of the ribonuclease barnase has been determined by using 1H nuclear magnetic resonance (NMR) spectroscopy. The 20 structures were calculated by using 853 interproton distance restraints obtained from analyses of two-dimensional nuclear Overhauser spectra, 72 phi and 53 chi 1 torsion angle res...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00099a030
更新日期:1991-09-03 00:00:00
abstract::Conceptually one may envisage that substrate binding sites on the ABC transporter P-gp cycle between high- and low-affinity conformations in response to signals arising from nucleotide hydrolysis to effect active transport. A radioligand binding assay was used to characterize the interaction of [3H]vinblastine with P-...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi011211z
更新日期:2001-12-25 00:00:00
abstract::Previously we showed that the low-molecular-weight mucin (MG2, encoded by MUC7), a major component of human submandibular/sublingual saliva, is a bacterial receptor that coats the tooth surface. Here we tested the hypothesis that the structure of its carbohydrate residues contains important information about its funct...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi972612a
更新日期:1998-04-07 00:00:00
abstract::The bifunctional reagent p-[(bromoacetyl)-amino]phenyl arsenoxide (BrCH2CONHPhAsO) in the presence of excess reduced nicotinamide adenine dinucleotide has been shown to cause the irreversible active site directed inactivation of the lipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase multienzyme (PD) ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00515a018
更新日期:1981-06-09 00:00:00
abstract::The crystal structure of the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) has been reported recently [Lieberman, R. L., and Rosenzweig, A. C. (2005) Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane, Nature 434, 177-182]. Subsequent ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi700883g
更新日期:2007-12-04 00:00:00
abstract::In spite of a nuclear targeting sequence, a substantial amount of dual-specificity tyrosine phosphorylation-regulated kinase (Dyrk1A) is located within the cytoplasm of neurons. Analysis of fractionated rat brains revealed that the majority of Dyrk1A was in the postnuclear precipitate. The kinase in this fraction was ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9010557
更新日期:2009-10-06 00:00:00
abstract::We have analyzed the role of several protein motifs in controlling the membrane association of the xlcaax-1 protein in Xenopus oocytes. Xlcaax-1 is a maternally expressed protein that during development is associated with the basal lateral membrane of polarized epithelial cells. It is enriched in the tubule cells of t...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00083a022
更新日期:1993-08-17 00:00:00
abstract::We have studied the Cu(II), Co(II), and Fe(III) complexes of the antineoplastic drug bleomycin by using electron spin--echo envelope spectroscopy. For all three complexes, nitrogen coordination of the metal ions is demonstrated. For the Cu(II)-- and Co(II)--drug complexes, we have been able to identify imidazole as a ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00509a045
更新日期:1981-03-17 00:00:00
abstract::Prolyl hydroxylase is a glycoprotein containing two nonidentical subunits, alpha and beta. The alpha subunit of prolyl hydroxylase isolated from 13-day-old chick embryos contains a single high mannose oligosaccharide having seven mannosyl residues. Two forms of alpha subunit have been shown to exist in enzyme purified...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00342a041
更新日期:1985-10-08 00:00:00
abstract::Segregation of mRNAs in the cytoplasm of polar cells has been demonstrated for proteins involved in Xenopus and Drosophila oogenesis, and for some proteins in somatic cells. It is assumed that vectorial transport of the messages is generally responsible for this localization. The mRNA encoding the basic protein of cen...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9800247
更新日期:1998-05-12 00:00:00
abstract::Hydrogen bonds which form between a hydrogen bond donor and an aromatic ring as acceptor are thought to contribute to the stability and function of proteins. We have tested the function of such an interaction in a highly homologous pair of proteins, cellular retinol-binding protein (CRBP) and cellular retinol-binding ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00035a018
更新日期:1995-09-05 00:00:00
abstract::Probing and interrogating protein interactions that involve acyl carrier proteins (ACP's) in fatty acid synthases and polyketide synthases are critical to understanding the molecular basis for the programmed assembly of complex natural products. Here, we have used unnatural amino acid mutagenesis to site specifically ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi500936u
更新日期:2014-12-09 00:00:00
abstract::A new bifunctional spin-label (BSL) has been synthesized that can be immobilized on the surface of proteins, allowing measurement of rotational motion of proteins by saturation-transfer electron paramagnetic resonance (STEPR). The spin-label contains a photoactivatable azido moiety, a cleavable disulfide, and a nitrox...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00476a014
更新日期:1990-06-19 00:00:00
abstract::We have examined the ability of the Escherichia coli single-stranded DNA binding protein (SSB) tetramer to form its different binding modes on poly(dC), poly(U), and poly(A) over a range of NaCl and NaF concentrations for comparison with previous studies with poly(dT). In reverse titrations with poly(U) and poly(A) at...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00186a016
更新日期:1994-05-24 00:00:00
abstract::Although point mutations usually lead to minor localized changes in protein structure, replacement of conserved Pro-76 with Gly in iso-2-cytochrome c induces a major conformational change. The change in structure results from mutation-induced depression of the pK for transition to an alkaline conformation with altered...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00451a043
更新日期:1989-12-12 00:00:00
abstract::The effect of mutating Gly 478 to Ala in rat cytochrome P450 2B1 on the metabolism of N-benzyl-1-aminobenzotriazole was investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of the wild-type enzyme was completely inactivated by incubating with 1 microM BBT. The G478A mutant, however, was not i...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi971064y
更新日期:1997-09-30 00:00:00
abstract::Calf brain tubulin was subjected to isoelectric focusing and tryptic peptide map analysis. Results from isoelectric focusing experiments showed a total number of 17 well-resolved protein peaks. The number of peaks and the mass distribution under each peak remained the same when the concentration of protein or ampholyt...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00512a006
更新日期:1981-04-28 00:00:00
abstract::Glutathione S-transferase, from the malarial parasite Plasmodium falciparum (PfGST), exerts a protective role in the organism and is thus considered an interesting target for antimalarial drug development. In contrast to other GSTs, it is present in solution as a tetramer and a dimer in equilibrium, which is induced b...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi400007g
更新日期:2013-03-19 00:00:00
abstract::Light-induced GTP-dependent scattering changes are studied in suspensions of retinal disc membranes to which one or both of the purified proteins involved in the phototransduction mechanism (G-protein and cGMP phosphodiesterase) are reassociated; a scattering change which depends on the presence of both G-protein (G) ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00121a039
更新日期:1992-02-18 00:00:00
abstract::The calcitonin/CGRP gene pre-mRNA is alternatively spliced in a tissue-specific manner resulting in the formation of calcitonin mRNA in thyroid C-cells and CGRP mRNA in neurons. Computer analysis of the RNA containing the 3' splice acceptor of the calcitonin-specific exon 4 predicts that this region has the potential ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9808058
更新日期:1998-11-10 00:00:00
abstract::The structure of cytochrome f includes an internal chain of five water molecules and six hydrogen-bonding side chains, which are conserved throughout the phylogenetic range of photosynthetic organisms from higher plants, algae, and cyanobacteria. The in vivo electron transfer capability of Chlamydomonas reinhardtii cy...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0004596
更新日期:2000-08-08 00:00:00
abstract::Isopentenyl diphosphate:dimethylallyl diphosphate isomerase (EC 5.3.3.2) catalyzes the antarafacial [1.3] allylic rearrangement of isopentenyl diphosphate (IPP) to its electrophilic allylic isomer dimethylallyl diphosphate (DMAPP). Active-site thiols at C138 and C139 were recently identified by covalent modification u...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00180a014
更新日期:1994-04-12 00:00:00
abstract::The heme-regulated inhibitor (HRI) of protein synthesis becomes activated in rabbit reticulocyte lysates in response to a variety of conditions including heme-deficiency, addition of oxidants, and heat shock. Activated HRI inhibits translation by catalyzing the phosphorylation of the alpha-subunit of eukaryotic initia...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00080a001
更新日期:1993-07-27 00:00:00
abstract::The binding modes of four active site-directed, acylating inhibitors of human alpha-thrombin have been determined using X-ray crystallography. These inhibitors (GR157368, GR166081, GR167088, and GR179849) are representatives of a series utilizing a novel 5, 5-trans-lactone template to specifically acylate Ser195 of th...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9830359
更新日期:1999-06-22 00:00:00
abstract::Induced fit has been postulated to be an important component of ligand interactions with proteins, including protein-DNA interactions. We imagined that the entropic cost of induced fit might be highly dependent on the local protein sequence context around critical contact residues. To investigate this question, we ana...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi960256a
更新日期:1996-06-25 00:00:00