Abstract:
:The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli catalyzes the oxidation of proline to glutamate in two reaction steps using separate proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase domains. Here, the kinetic mechanism of PRODH in PutA is studied by stopped-flow kinetics to determine microscopic rate constants for the proline:ubiquinone oxidoreductase mechanism. Stopped-flow data for proline reduction of the flavin cofactor (reductive half-reaction) and oxidation of reduced flavin by CoQ(1) (oxidative half-reaction) were best-fit by a double exponential from which maximum observable rate constants and apparent equilibrium dissociation constants were determined. Flavin semiquinone was not observed in the reductive or oxidative reactions. Microscopic rate constants for steps in the reductive and oxidative half-reactions were obtained by globally fitting the stopped-flow data to a simulated mechanism that includes a chemical step followed by an isomerization event. A microscopic rate constant of 27.5 s(-1) was determined for proline reduction of the flavin cofactor followed by an isomerization step of 2.2 s(-1). The isomerization step is proposed to report on a previously identified flavin-dependent conformational change [Zhang, W. et al. (2007) Biochemistry 46, 483-491] that is important for PutA functional switching but is not kinetically relevant to the in vitro mechanism. Using CoQ(1), a soluble analogue of ubiquinone, a rate constant of 5.4 s(-1) was obtained for the oxidation of flavin, thus indicating that this oxidative step is rate-limiting for k(cat) during catalytic turnover. Steady-state kinetic constants calculated from the microscopic rate constants agree with the experimental k(cat) and k(cat)/K(m) parameters.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Moxley MA,Becker DFdoi
10.1021/bi201603fsubject
Has Abstractpub_date
2012-01-10 00:00:00pages
511-20issue
1eissn
0006-2960issn
1520-4995journal_volume
51pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::Purple membranes (PM) from Halobacterium halobium were incorporated into 7.5% polyacrylamide gels to prevent aggregation which occurs in suspensions at low pH. At pH 7.0, the circular dichroism (CD) spectra and visible absorption spectra of light- and dark-adapted bacteriorhodopsin (bR558, respectively) and the flash ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00586a007
更新日期:1979-09-18 00:00:00
abstract::In fosfomycin biosynthesis, the hydrolysis of cytidylyl ( S)-2-hydroxypropylphosphonate [( S)-HPP-CMP] to afford ( S)-HPP is the only uncharacterized step. Because FomD is an uncharacterized protein with a DUF402 domain that is encoded in the fosfomycin biosynthetic gene cluster, FomD was hypothesized to be responsibl...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.8b00690
更新日期:2018-08-14 00:00:00
abstract::Porcine parathyroid gland slices were incubated with 3H-labeled amino acids in order to label tissue proteins. After incubation a crude hormonal extract was prepared and analyzed by chromatography on carboxymethylcellulose. Among the three radioactive peaks which were detected in the eluate, two were identified as par...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00687a018
更新日期:1975-08-12 00:00:00
abstract::A new approach to studying membrane topology and permissive sites in membrane proteins expressed in Escherichia coli is described. The method is based on in vitro transposition of mini-Tn5 derivatives bearing dual pho-lac reporters [Alexeyev, M. F., and Winkler, H. H. (1999) J. Mol. Biol. 285, 1503-1513]. Two mini-Tn5...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi011813i
更新日期:2002-01-08 00:00:00
abstract::The linear noncompetitive inhibition of the pepsin-catalyzed hydrolysis of Ac-Phe-Phe-Gly at pH 2.1 by L-Ac-Phe, L-Ac-Phe-NH2, and L-Ac-Phe-OEt has been claimed to substantiate the ordered release of products specified by the amino-enzyme mechanism for pepsin action. According to this interpretation, the binding of in...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00674a023
更新日期:1975-02-11 00:00:00
abstract::A core Y61F mutant of the gene 5 single-stranded DNA-binding protein (g5p) of f1 bacterial virus aggregated when expressed from a plasmid, but, after refolding in vitro, it behaved much like wild-type and may be a stability or folding mutant. Circular dichroism (CD) titrations showed the same cooperative polynucleotid...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi972545k
更新日期:1998-05-19 00:00:00
abstract::Atlantic salmon fast skeletal muscle is composed of an α-type tropomyosin that shares 20 substitutions with a mammalian homologue. Prominent isomorphisms considered to be potentially destabilizing include threonine 179 (core, Ala in rabbit) and a unique pair of glycines, residues 24 and 27. Bacterially expressed mutan...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.5b00156
更新日期:2015-05-05 00:00:00
abstract::N-[[(Iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS) is a fluorescent reagent which reacts covalently with the free thiol groups of proteins. When the reagent is reacted with the Escherichia coli ribosome under mild conditions, gel electrophoresis shows modification of predominantly two proteins, S18...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00346a042
更新日期:1985-12-03 00:00:00
abstract::Phospholipid transfer protein (PLTP) is expressed by macrophage-derived foam cells in human atherosclerotic lesions, suggesting a regulatory role for PLTP in cellular cholesterol homeostasis. However, the exact role of PLTP in the reverse cholesterol transport pathway is not known. PLTP is present in plasma as two for...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi700833h
更新日期:2007-10-23 00:00:00
abstract::Tyrosine hydroxylase (TH) catalyzes the first step in the biosynthesis of catecholamines. Regulation of TH enzyme activity is controlled through the posttranslational modification of its regulatory domain. The regulatory domain of TH can be phosphorylated at four serines (8, 19, 31, and 40) by a variety of protein kin...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi7019468
更新日期:2008-02-12 00:00:00
abstract::We have characterized the importance of size, sulfation, and anticoagulant activity of heparin in release of basic fibroblast growth factor (bFGF) from the subendothelial extracellular matrix (ECM) and the luminal surface of the vascular endothelium. For this purpose, 125I-bFGF was first incubated with ECM and conflue...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00122a027
更新日期:1992-02-25 00:00:00
abstract::The malarial parasite Plasmodium falciparum can only synthesize pyrimidine nucleotides via the de novo pathway which is therefore a suitable target for development of antimalarial drugs. New assay procedures have been developed using high-pressure liquid chromatography (HPLC) which enable concurrent measurement of pyr...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00183a033
更新日期:1994-05-03 00:00:00
abstract::The acetoacetate decarboxylase-like superfamily (ADCSF) is a little-explored group of enzymes that may contain new biocatalysts. The low level of sequence identity (∼20%) between many ADCSF enzymes and the confirmed acetoacetate decarboxylases led us to investigate the degree of diversity in the reaction and substrate...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.9b00652
更新日期:2019-10-08 00:00:00
abstract::A series of cysteine-bearing hydrophobic polypeptides analogous to a light-harvesting one betapolypeptide (LH1beta) from the LH1 complex from the purple photosynthetic bacterium, Rhodobacter sphaeroides, was synthesized using an Escherichia coli expression system. The cysteine was placed in the C- or N-terminal region...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0478493
更新日期:2005-04-05 00:00:00
abstract::The sequential solid-phase synthesis of a peptide analogue of bovine brain calmodulin calcium binding site III covering residues 81-113 of the natural sequence is described. Methionine-109 is replaced by a leucine residue to avoid complications in the synthesis and purification. In an attempt to relate the structure o...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00393a019
更新日期:1987-09-22 00:00:00
abstract::The epidermal growth factor receptor (EGFR) kinase is activated by a variety of mutations in human cancers. R776H is one such recurrent mutation (R752H in another numbering system) in the αC-β4 loop of the tyrosine kinase domain that activates EGFR in the absence of the activating EGF ligand. However, the mechanistic ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.5b00444
更新日期:2015-07-14 00:00:00
abstract::Dihydrofolate and dihydropteroylpolyglutamates inhibit pig liver methylenetetrahydrofolate reductase. In all cases the inhibition is linearly competitive with respect to methylenetetrahydrofolate. The Ki values decrease with each additional glutamyl residue from one to six, from a value of 6.5 microM for dihydrofolate...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00551a005
更新日期:1980-05-13 00:00:00
abstract::Ultraviolet (uv)-light-mediated formation of protein-DNA adducts in Chinese hamster cell chromatin was investigated in an attempt to compare chromatin alterations induced in vitro with those observed in vivo. Three independent methods of analysis indicated stable protein-DNA associations: (1) a membrane filter assay w...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00653a019
更新日期:1976-04-20 00:00:00
abstract::The insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) is a type I glycoprotein that mediates both the intracellular sorting of lysosomal enzymes bearing mannose 6-phosphate (Man-6-P) residues to the lysosome and the bioavailability of IGF-II. The extracytoplasmic region of the IGF-II/MPR contains...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi992226o
更新日期:2000-03-07 00:00:00
abstract::T4 RNA ligase catalyzes the ATP-dependent addition of a single 2'-deoxyribonucleoside 3',5'-bisphosphate to the 3'-hydroxyl of an oligodeoxyribonucleotide. The bisphosphate is joined to the deoxyoligomer by a 3' leads to 5' phosphodiester bond and the product, which is terminated by a 3'-phosphate, is one nucleotide l...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00617a004
更新日期:1978-11-28 00:00:00
abstract::The assignment of disulfide bonds in human J chain and its covalent pairing with immunoglobulin M was determined under conditions which minimize disulfide bond interchange. We show that in J chain the three intradisulfide bridges are formed between Cys 12 and 100, Cys 71 and 91, and Cys 108 and 133. Previous reports [...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00165a014
更新日期:1992-12-22 00:00:00
abstract::Preferential interaction of trans-membrane alpha-helices whose hydrophobic length matches the hydrophobic thickness of the lipid bilayer could be a mechanism of retention in the Golgi apparatus. We have used fluorescence methods to study the interaction of peptides Ac-K2-G-Lm-W-Ln-K2-A-amide (Pm+n) with bilayers of ph...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi972441+
更新日期:1998-01-13 00:00:00
abstract::The human endonuclease III homologue (hNTH1) removes premutagenic cytosine damage from DNA. This includes 5-hydroxycytosine, which has increased potential for pairing with adenine, resulting in C --> T transition mutations. Here we report that hNTH1 acts on both 5-hydroxycytosine and abasic sites preferentially when t...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0028901
更新日期:2001-06-05 00:00:00
abstract::HTC cells have been labeled by short exposures to [3H]thymidine in order to identify newly synthesized DNA. By either isolating nuclei directly or isolating them after an extensive fixation with formaldehyde, we have been able to identify two phases in the maturation process of newly replicated chromatin. The first ph...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00302a036
更新日期:1984-03-27 00:00:00
abstract::Type IIS restriction endonuclease Eco31I harbors a single HNH active site and cleaves both DNA strands close to its recognition sequence, 5'-GGTCTC(1/5). A two-domain organization of Eco31I was determined by limited proteolysis. Analysis of proteolytic fragments revealed that the N-terminal domain of Eco31I is respons...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi800660u
更新日期:2008-08-19 00:00:00
abstract::The left-handed polyproline II (P(II)) helix is a structure that has been given a great deal of attention lately because of its role in a wide variety of physiologically important processes and potential significance in protein unfolded states. Recent work by several authors has shown that residues besides proline can...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi049922v
更新日期:2004-05-18 00:00:00
abstract::The amidotransferase family of enzymes utilizes the ammonia derived from the hydrolysis of glutamine for a subsequent chemical reaction catalyzed by the same enzyme. The ammonia intermediate does not dissociate into solution during the chemical transformations. A well-characterized example of the structure and mechani...
journal_title:Biochemistry
pub_type: 杂志文章,评审
doi:10.1021/bi990871p
更新日期:1999-06-22 00:00:00
abstract::Vitronectin is a multifunctional plasma glycoprotein which may regulate the systems related to protease cascades such as the coagulation, fibrinolysis, and complement systems as well as cell adhesion. Solid-phase assays and affinity chromatography on immobilized glycolipids indicated that vitronectin purified under de...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi972247n
更新日期:1998-05-05 00:00:00
abstract::Ionizing radiation, free radicals, and reactive oxygen species produce hundreds of different DNA lesions. Clustered lesions are typical for ionizing radiation. They compromise the efficiency of the base excision repair (BER) pathway, and as a consequence, they are much more toxic and mutagenic than isolated lesions. D...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.6b00396
更新日期:2016-07-19 00:00:00
abstract::An S3 electron paramagnetic resonance (EPR) signal is observed in a variety of photosystem 2 (PS2) samples in which the oxygen-evolving complex (OEC) has been inhibited. These signals have been proposed to be due to an interaction, S2X+, between the manganese cluster in an oxidation state equivalent to S2 and an organ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00088a032
更新日期:1993-09-21 00:00:00