Abstract:
:The residue C221 on pyruvate decarboxylase (EC. 4.1.1.1) from Saccharomyces cerevisiae has been shown to be the site where the substrate activation cascade is triggered [Baburina et al. (1994) Biochemistry 33, 5630-5635] and is located on the beta domain [Arjunan et al. (1996) J. Mol. Biol. 256, 590], while the active-center thiamin diphosphate is located > 20 A away, at the interface of the alpha and gamma domains. The reactivity of all three exposed cysteines (152, 221, and 222) was examined under the influence of known activators and inhibitors. Protein chemical methods, in conjunction with [1-14C] and [3-3H] analogues of the mechanism-based inhibitor p-ClC6H4CH=CHCOCOOH, demonstrated that the holoenzyme bound approximately 2-3 atoms of tritium/atom of C-14. However, when the labeled enzyme was subjected to trypsinization, followed by sequencing of the labeled peptide, only the tritium label was in evidence at C221, with a stoichiometry of 2 atoms of tritium/tetrameric holoenzyme. Apparently, the product of decarboxylation bonded to the enzyme survived the limited proteolysis and sequencing, but the bound 2-oxoacid was released during the protocol. Surprisingly, the C221S or C222A variants, although they still possess 20-30% specific activity compared to the wild-type enzyme, could still be inhibited by the XC6H4CH=CHCOCOOH class of inhibitors/substrate analogues, as well as by the product of decarboxylation from such compounds, cinnamaldehydes. Other potential nucleophilic sites for the inhibitor [C152 (the third exposed cysteine), residues D28, H114, H115, and E477 at the active center and H92 at the regulatory site] were also substituted by a nonnucleophilic side chain. All variants were still subject to inhibition by p-ClC6H4CH=CHCOCOOH, the active-center variants being inactivated even faster than the wild-type enzyme, suggesting that the active center is involved in the inactivation process. It appears that C221 is one of only two sites of interaction with such compounds (perhaps the result of a Michael addition across the C=C bond), yet the bound [1-14C]-labeled inhibitor could no longer be detected after peptide mapping at this site or at the catalytic site. Upon combining the tritiated inhibitor with [2-14C]-thiamin diphosphate, no evidence could be found for a thiamin-inhibitor-protein ternary complex, suggesting that the thiamin-bound enamine intermediate did not react further with the protein. It is likely that the second form of inhibition is at the active center, with the inhibitor cofactor-bound, which would have been released during the proteolytic protocol. Among other known activators, ketomalonate was found to react at C221 only. Glyoxalic acid, a mechanism-based inhibitor, on the other hand, could react at both the regulatory and the catalytic center. The high reactivity of C221 is consistent with it being in the thiolate form at the optimal pH of the enzyme [forming a Cys221S(-) + HHis92 ion pair; see Baburina et al. (1996) Biochemistry 35, 10249-10255, and Baburina et al. (1998) Biochemistry 37, 1235-1244]. Several additional compounds were tested as potential regulatory site-directed reagents: iodoacetate, 1,3-dibromoacetone, and 1-bromo-2-butanone. All three compounds reduced the Hill coefficient and hence appear to react at C221. It was concluded that either substitution of C221 by a nonnucleophilic residue or large groups attached to C221 in the wild-type enzyme lead to a distortion of domain interactions, interactions which are required for both optimal activity and substrate activation.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Baburina I,Dikdan G,Guo F,Tous GI,Root B,Jordan Fdoi
10.1021/bi9709912subject
Has Abstractpub_date
1998-02-03 00:00:00pages
1245-55issue
5eissn
0006-2960issn
1520-4995pii
bi9709912journal_volume
37pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::Several bifunctional alkylating agents of the aziridinylbenzoquinone class have been evaluated as potential antitumor agents. 3,6-Bis[(2-hydroxyethyl)amino]-2,5- diaziridinyl-1,4-benzoquinone (BZQ), 2,5-diaziridinyl-1,4-benzoquinone (DZQ), 3,6-bis(carboxyamino)-2,5-diaziridinyl- 1,4-benzoquinone (AZQ), and six analogu...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00114a016
更新日期:1991-12-17 00:00:00
abstract::Conformations of (R)-alpha-(N6-adenyl)styrene oxide adducts at positions X6 in d(CGGACXAGAAG).d(CTTCTTGTCCG) and X7 in d(CGGACAXGAAG).d(CTTCTTGTCCG), incorporating codons 60, 61 (underlined), and 62 of the human n-ras protooncogene, were refined from 1H NMR data. These were the R(61,2) and R(61,3) adducts. Chemical sh...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00043a008
更新日期:1995-10-31 00:00:00
abstract::Polyisoprenyl diphosphates play diverse and vital roles in cell function in health and disease. The counter-regulatory lipid signaling molecule, presqualene diphosphate (PSDP), is rapidly converted to its monophosphate form (PSMP) upon cell activation [Levy, B. D., Petasis, N. A., and Serhan, C. N. (1997) Nature 389, ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi8020636
更新日期:2009-04-07 00:00:00
abstract::The low level of enzymatic activity of certain alpha 2-macroglobulin-proteinase complexes could be important to the function of factor VIII/von Willebrand glycoprotein since it is especially sensitive to proteolytic cleavage. To test this possibility, complexes of alpha 2-macroglobulin with plasmin, trypsin, and throm...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00275a018
更新日期:1983-03-15 00:00:00
abstract::DNA containing short sequences of the form (dA)n.(dT)n can exhibit pronounced degrees of stable curvature of the helix axis, provided that these homooligomeric stretches are approximately in phase with the helix repeat. However, the precise origin of this effect is unknown. We have observed that pyrimidine 5-methyl gr...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00460a003
更新日期:1990-02-27 00:00:00
abstract::The crystal structure of an extracellular triglyceride lipase (from a fungus Rhizomucor miehei) inhibited irreversibly by diethyl p-nitrophenyl phosphate (E600) was solved by X-ray crystallographic methods and refined to a resolution of 2.65 A. The crystals are isomorphous with those of n-hexylphosphonate ethyl ester/...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00120a034
更新日期:1992-02-11 00:00:00
abstract::Resonance Raman spectra of squid rhodopsin have been obtained under a variety of temperature and illumination conditions. The data have been characterized in terms of spectral contributions from squid rhodopsin, isorhodopsin, bathorhodopsin, lumirhodopsin, mesorhodopsin, P-465, and acid metarhodopsin. The results are ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00615a018
更新日期:1978-10-31 00:00:00
abstract::Halogen bonding is a weak chemical force that has so far mostly found applications in crystal engineering. Despite its potential for use in drug discovery, as a new molecular tool in the direction of molecular recognition events, it has rarely been assessed in biopolymers. Motivated by this fact, we have developed a p...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.7b00429
更新日期:2017-06-27 00:00:00
abstract::The X-ray crystal structures of the complexes of the recombinant kringle 1 domain of human plasminogen (Klpg) with the ligands epsilon-aminocaproic acid (EACA) and trans-4-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA), which are representative of a class of in vivo antifibrinolytic agents, have been determined at...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9521351
更新日期:1996-02-27 00:00:00
abstract::We have synthesized 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(N-oleoyl)serine (N-acyl-PS) and 1,2-dioleoyl-sn-glycero-3-phospho-Tris (phosphatidyl-Tris) and have characterized both phospholipids by their chemical and chromatographic properties, as well as by their IR, 13C NMR, and 1H NMR spectra. Comparison of these d...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00221a003
更新日期:1991-02-19 00:00:00
abstract::We have expressed the alpha and beta subunits of bacterial luciferase, encoded by luxA and luxB, from separate plasmids in Escherichia coli and developed an efficient purification scheme that yields many milligrams of protein of greater than 90% homogeneity. Earlier experiments showed that subunits synthesized separat...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00070a010
更新日期:1993-05-18 00:00:00
abstract::Bactenecin 5 and its fragments [BN22 (1-22), BN16 (7-22), and BC24 (20-43)] were synthesized by solid-phase methods. Their antifungal activities on Candida albicans have been studied and compared with those of the native bactenecin 5. The conformational preferences of these peptides in aqueous and nonaqueous solutions...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi951681r
更新日期:1996-04-09 00:00:00
abstract::Creatininase catalyzes the conversion of creatinine (a biosensor for kidney function) to creatine via a two-step mechanism: water addition followed by ring opening. Water addition is common to other known cyclic amidohydrolases, but the precise mechanism for ring opening is still under debate. The proton donor in this...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.7b01032
更新日期:2017-12-05 00:00:00
abstract::Amphiphiles respond both to polar and to nonpolar solvents. In this paper X-ray diffraction and osmotic stress have been used to examine the phase behavior, the structural dimensions, and the work of deforming the monolayer-lined aqueous cavities formed by mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleo...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00453a010
更新日期:1990-01-09 00:00:00
abstract::Both activities of the aspartokinase--homoserine I (AK-HSD) of Escherichia coli are inhibited by threonine. Careful threonine binding studies have now been done which have allowed us to distinguish the various effects of threonine on the enzyme. The ultrafiltration technique of H. Paulus ((1969) Anal. Biochem. 32, 101...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00610a014
更新日期:1978-08-22 00:00:00
abstract::Phase partition techniques have been used to measure the binding of the antitumor drugs adriamycin (NSC-123127) and daunorubicin (NSC-82151) to various DNAs. These methods provide reliable equilibrium binding data at the low levels of drug binding that may be expected in vivo. Both adriamycin and daunorubicin exhibit ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00285a033
更新日期:1983-08-02 00:00:00
abstract::The dependence of enzymatic catalysis on diffusion rates in solution was examined with regard to high specific activity carbonic anhydrase (CA II) by varying the viscosity of the reaction medium with added glycerol, sucrose, and ficoll (a copolymer of sucrose and epichlorohydrin). Responses of the Michaelis-Menten par...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00383a028
更新日期:1987-05-05 00:00:00
abstract::We have performed comparative studies of the neutral glycosphingolipids synthesized by three human myeloid leukemia cell lines, K562, KG1, and HL-60, which were metabolically labeled with [14C]galactose, to evaluate changes in neutral glycosphingolipid synthesis with myeloid cell differentiation. Individual neutral gl...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00345a034
更新日期:1985-11-19 00:00:00
abstract::After extensive studies spanning over half a century, there is little consensus on the kinetic mechanism of DNA polymerases. Using stopped-flow fluorescence assays for mammalian DNA polymerase beta (Pol beta), we have previously identified a fast fluorescence transition corresponding to conformational closing, and a s...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi700084w
更新日期:2007-05-08 00:00:00
abstract::A series of secreted proteases are included among the virulence factors documented for Staphylococcus aureus. In light of increasing antibiotic resistance of this dangerous human pathogen, these proteases are considered as suitable targets for the development of novel therapeutic strategies. The recent discovery of st...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi035310j
更新日期:2003-11-25 00:00:00
abstract::We have found that the atomic force microscope (AFM) can be used to image the "beads-on-a-string" chromatin structure in a normal air environment following adsorption onto a cover glass substrate. Individual nucleosome cores and linker DNA could be resolved clearly along chromatin fibers that were reconstituted using ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00084a002
更新日期:1993-08-24 00:00:00
abstract::Rhodospirillum rubrum ribulose bisphosphate carboxylase contains two high affinity binding sites for pyridoxal phosphate and two catalytic sites per dimer. However, pyridoxal phosphate binding at only one site is sufficient for inactivation of both catalytic sites. In the presence of 20 mM bicarbonate, 10 mM magnesium...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00600a024
更新日期:1978-04-04 00:00:00
abstract::The neutral glycosphingolipids of hairy cells from a patient with hairy cell leukemia were chemically analyzed by thin-layer and gas-liquid chromatography, mass spectrometry, combined gas chromatography-mass spectrometry, and glycosidase treatment. These cells were found to have compounds containing one to four sugars...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00525a032
更新日期:1981-10-27 00:00:00
abstract::The structural determinants of substrate specificity of human liver cytochrome P450 2C8 (CYP2C8) were investigated using site-directed mutants chosen on the basis of a preliminary substrate pharmacophore and a three-dimensional (3D) model. Analysis of the structural features common to CYP2C8 substrates exhibiting a mi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0489309
更新日期:2004-12-14 00:00:00
abstract::The temperature dependence of the packing (order) and fluidity (microviscosity) of rabbit small, intestinal brush border vesicle membranes and of liposomes made from their extracted lipids has been investigated by using a variety of lipid spin probes. The lipids in the brush border membrane are present essentially as ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00265a036
更新日期:1982-10-26 00:00:00
abstract::Oxidation of low-density lipoprotein (LDL), the major cholesterol carrier in plasma, is thought to promote atherogenesis via several mechanisms. One proposed mechanism involves fusion of oxidized LDL in the arterial wall; another involves oxidation-induced amyloid formation by LDL apolipoprotein B. To test these mecha...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi700225a
更新日期:2007-05-15 00:00:00
abstract::The major carbohydrate fragment from the lipophosphoglycan of Leishmania donovani was generated by mild acid hydrolysis (0.02 N HCl, 5 min, 100 degrees C) and purified by chromatography on DE-52 cellulose and thin layer. By a combination of analyses including gas-liquid chromatography-mass spectrometry and 1H NMR, the...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00393a042
更新日期:1987-09-22 00:00:00
abstract::The inhibitory effects of urea on the normally rapid fibrillation of human calcitonin (hCT) were investigated by 1H NMR. From subtle differences in the chemical shift of hCT in the presence and absence of urea, the occurrence of weak interactions between urea and hCT was confirmed. The chemical shifts on the NH and C ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi961013l
更新日期:1996-10-01 00:00:00
abstract::A novel truncated form (residues 1-214, with a randomized C-terminal tail) of the ligand-binding extracellular domain (ECD) of the human alpha1 glycine receptor (GlyR), with amino acids from the corresponding sequence of an acetylcholine binding protein (AChBP) substituted for two relatively hydrophobic membrane-proxi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi800659x
更新日期:2008-09-16 00:00:00
abstract::It is known that, while melittin at micromolar concentrations is unfolded under conditions of low ionic strength at neutral pH, it adopts a tetrameric alpha-helical structure under conditions of high ionic strength, at alkaline pH, or at high peptide concentrations. To understand the mechanism of the conformational tr...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00118a014
更新日期:1992-01-28 00:00:00