Abstract:
:The enzyme kinetics of the reduction of the substrate 6,8-dimethylpterin by chicken and recombinant human dihydrofolate reductases (DHFRs) have been examined over the pH range 5.0-8.0 in the presence of NADPH or (4R)-[2H]NADPH (NADPD). The pH profiles of the catalytic constant (Vmax/[E]o or kcat) showed pH independence for chicken DHFR and little pH dependence for human DHFR. For both DHFRs, the pH profiles of the Michaelis constant (Km(substrate)) and the apparent second-order rate constant (Vmax/Km(substrate)[E]o or kcat/Km(substrate)) indicated that two ionizable groups, deduced to be the substrate and the conserved Glu carboxy group in the enzyme active site, should be ionized in their cationic and anionic forms, respectively, for formation of the enzyme-substrate complex and for catalysis. The pKa values of about 5.3 and 6.5 which were obtained from the pH profiles of Km(substrate) and kcat/Km(substrate) were assigned to the ionizations of the substrate and the enzyme carboxy group, respectively. Deuterium isotope effects on DV and d(V/K) were significant for both enzymes, approximately 3 for chicken DHFR and approximately 4 for recombinant human DHFR, and were pH independent. Thus, the rate-limiting step in the enzymic reduction of 6,8-dimethylpterin is hydride-ion transfer at acidic pHs as well as neutral pHs. The results demonstrate that, compared with dihydrofolate, 6,8-dimethylpterin is a superior substrate for mechanistic investigations as it allows direct study of the effects of both enzyme and substrate ionizations involved in the catalysis and also avoids the obscuration of the catalytic rate by product release.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Jeong SS,Gready JEdoi
10.1021/bi00011a031subject
Has Abstractpub_date
1995-03-21 00:00:00pages
3734-41issue
11eissn
0006-2960issn
1520-4995journal_volume
34pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::Endonuclease V of bacteriophage T4 binds to UV-irradiated deoxyribonucleic acid (DNA) but not to unirradiated DNA. We have developed an assay to detect this binding, based on the retention of enzyme--DNA complexes on nitrocellulose filters. The amount of complex retained, ascertained by using radioactive DNA, is a mea...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00549a026
更新日期:1980-04-15 00:00:00
abstract::The fluorescent pH indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to investigate changes in the intracellular pH (pHi) of individual Sf9 cells in response to changes in the composition of the external medium. Under standard conditions, the resting pHi was 0.2-0.3 unit lower than the extracel...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00046a023
更新日期:1995-11-21 00:00:00
abstract::Gap junctions, transmembrane protein channels that directly connect the cytoplasm of neighboring cells and enable the exchange of molecules between cells, are a promising new frontier for therapeutic delivery. Specifically, cell-derived lipid vesicles that contain functional gap junction channels, termed Connectosomes...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.7b00688
更新日期:2018-01-09 00:00:00
abstract::Further to our studies on the Escherichia coli 30S ribosomal subunit, the detailed surface topography of both 50S subunits and 70S ribosomes has been investigated by using iodination catalyzed by immobilized lactoperoxidase as the surface probe. In the 50S subunit, only proteins L2, L5, L10, and L11 were iodinated to ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00282a019
更新日期:1983-06-21 00:00:00
abstract::Currently, it is thought that inhalational anesthetics cause anesthesia by binding to ligand-gated ion channels. This is being investigated using four-alpha-helix bundles, small water-soluble analogues of the transmembrane domains of the "natural" receptor proteins. The study presented here specifically investigates h...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi034623b
更新日期:2003-09-30 00:00:00
abstract::The unique characteristics of DNA charge transport (CT) have prompted an examination of roles for this chemistry within a biological context. Not only can DNA CT facilitate long-range oxidative damage of DNA, but redox-active proteins can couple to the DNA base stack and participate in long-range redox reactions using...
journal_title:Biochemistry
pub_type: 杂志文章,评审
doi:10.1021/bi501520w
更新日期:2015-02-03 00:00:00
abstract::2-Azidoadenosine 5'-diphosphate (2-azido-ADP) labeled with 32P in the alpha-position was prepared and used to photolabel the nucleotide binding sites of beef heart mitochondrial F1-ATPase. The native F1 prepared by the procedure of Knowles and Penefsky [Knowles, A. F., & Penefsky, H. S. (1972) J. Biol. Chem. 247, 6617...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00346a052
更新日期:1985-12-03 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::A double mutant cycle has been used to evaluate interaction energies between the global stabilizer mutation asparagine 52 --> isoleucine (N52I) in iso-1-cytochrome c and mutations producing single surface histidines at positions 26, 33, 39, 54, 73, 89, and 100. These histidine mutation sites are distributed through th...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi034958t
更新日期:2003-09-16 00:00:00
abstract::Glutaminase (GLS1/2) catalyzes the conversion of L-glutamine to L-glutamate and ammonia. The level of a splice variant of GLS1 (GAC) is elevated in certain cancers, and GAC is specifically inhibited by bis-2-(5-phenylacetimido-1,2,4,thiadiazol-2-yl)ethyl sulfide (BPTES). We report here the first full-length crystal st...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi201613d
更新日期:2011-12-20 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::We show that eight different serine proteinases--bovine chymotrypsins A and B, porcine pancreatic elastase I, proteinase K, Streptomyces griseus proteinases A and B, and subtilisins BPN' and Carlsberg--interact with turkey ovomucoid third domain at the same Leu18-Glu19 peptide bond, the reactive site of the inhibitor....
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00341a007
更新日期:1985-09-24 00:00:00
abstract::CDPKs are a family of calcium (Ca2+)-dependent protein kinases which are defined by a carboxyl-terminal calmodulin-like domain. Mutational analysis indicates that the junction domain, which joins the kinase and calmodulin-like domains, contains an autoinhibitor. CDPK isoform AK1 from Arabidopsis was expressed in Esche...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00189a031
更新日期:1994-06-14 00:00:00
abstract::Phosphoinositide 3-kinase gamma (PI3Kgamma) is activated by Gbetagamma release after stimulation of Galpha i -coupled receptors, involving a recruitment of the enzyme to the plasma membrane via interaction of the regulatory subunit p101 or p87 with Gbetagamma. The receptor-mediated release of Gbetagamma was, however, ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi800596b
更新日期:2008-10-28 00:00:00
abstract::Among hormone-inducible transcription factors, estrogen receptors (ERs) play important roles in tissue growth and differentiation, via either direct or indirect binding, in the nucleus, to specific DNA targets called estrogen responsive elements (EREs), or through nongenomic pathways. In humans, two estrogen receptor ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.5b00026
更新日期:2015-06-30 00:00:00
abstract::The substrate specificities of two incorrectly annotated enzymes belonging to cog3964 from the amidohydrolase superfamily were determined. This group of enzymes are currently misannotated as either dihydroorotases or adenine deaminases. Atu3266 from Agrobacterium tumefaciens C58 and Oant2987 from Ochrobactrum anthropi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi301483z
更新日期:2013-01-08 00:00:00
abstract::Apolipophorin III (apoLp-III) from the greater wax moth Galleria mellonella is an exchangeable insect apolipoprotein that consists of five amphipathic alpha-helices, sharing high sequence identity with apoLp-III from the sphinx moth Manduca sexta whose structure is available. To define the minimal requirement for apoL...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi0013804
更新日期:2001-03-13 00:00:00
abstract::Ligand-induced dimerization of receptor tyrosine kinases (RTKs) modulates a system of linked biochemical reactions, sharply switching the RTK from a quiescent state to an active state that becomes phosphorylated and triggers intracellular signaling pathways. To improve our understanding of this molecular switch, we de...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi701892f
更新日期:2008-04-01 00:00:00
abstract::Diffusion studies by photon correlation of scattered laser light confirm the dissociation of the tetrameric form of human carboxyhemoglobin to dimers above pH 10 and provide new estimates of the subunit dissociation equilibrium constants in this pH range. Electrophoretic light-scattering experiments under the same con...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00616a014
更新日期:1978-11-14 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::Phospholipids containing a saturated fatty acid in the primary position and an unsaturated fatty acid in the secondary position are a major structural part of biological membranes. The mixed-chain hydrophobic core of the membranes is the diacylglycerol part. To better understand the core properties of membranes we hav...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00051a015
更新日期:1995-12-26 00:00:00
abstract::Most terpene synthase reactions follow Markovnikov rules for formation of high-energy carbenium ion intermediates. However, there are notable exceptions. For example, pentalenene synthase (PS) undergoes an initial anti-Markovnikov cyclization reaction followed by a 1,2-hydride shift to form an intermediate humulyl cat...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.0c00518
更新日期:2020-09-08 00:00:00
abstract::Vibrational circular dichroism (VCD) and Fourier transform IR (FTIR) were measured for a series of short alanine-based peptides having the general formula Ac-(AAKAA)n-GY-NH2 (n = 1-4) from 5 to 50 degrees C in D2O and at room temperature in both TFE and H2O. In both of these latter solvents, the dominant structural fo...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi971460g
更新日期:1997-12-09 00:00:00
abstract::Two hybridomas producing monoclonal antibodies to poly(adenosine diphosphate ribose) [poly(ADP-Rib)] were established. One antibody, 10H (IgG3, kappa), bound to most of the poly(ADP-Rib) preparation, which consisted of molecules of various sizes of more than 20 ADP-Rib residues. The binding of this antibody was inhibi...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00311a032
更新日期:1984-07-31 00:00:00
abstract::In this work, we show that adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) is a substrate for yeast inorganic pyrophosphatase (PPase) (EC 3.6.1.1) and further, using chirally labeled [gamma-17O,18O]ATP gamma S, that enzyme-catalyzed hydrolysis to produce chiral inorganic thio[17O,18O]phosphate proceeds with inversio...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00300a002
更新日期:1984-02-28 00:00:00
abstract::Single lamellar phosphatidyl[methyl-2H]choline vesicles were incubated with an excess of unlabeled phosphatidylcholine vesicles or phosphatidylcholine-cholesterol vesicles containing 8 mol % glucuronosyldiglyceride. Incubation of the two vesicle populations was performed in the presence or absence of a purified phosph...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00534a017
更新日期:1982-03-02 00:00:00
abstract::A specific, high-affinity binding site for ammodytoxin C in synaptic membranes from bovine cerebral cortex was detected and partially characterized. Equilibrium binding analysis revealed a single population of [125I]ammodytoxin C acceptors with the following binding parameters: Kd = 6.0 nM and Bmax = 5.7 pmol/mg membr...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00250a049
更新日期:1994-11-22 00:00:00
abstract::The precipitation of a monoclonal IgG2 crystalline cryoglobulin (WEB) is shown to be highly dependent on temperature and concentration. Below a critical concentration of 0.6 mg/mL there is no cryoprecipitation. The kinetics of the aggregation exhibits a concentration-dependent lag time. This evidence suggests that a n...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00579a027
更新日期:1979-06-12 00:00:00
abstract::The chicken and goose egg white lysozymes (ChEWL and GoEWL) are homologues, but differ in substrate specificity. ChEWL catalyzes the hydrolysis of the glycosidic bonds of bacterial peptidoglycans and chitin-derived substrates, while GoEWL is specific for bacterial peptidoglycans. The active-site aspartate 52 residue o...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi951671q
更新日期:1996-02-13 00:00:00
abstract::Expansions of (CTG)·(CAG) repeated DNAs are the mutagenic cause of 14 neurological diseases, likely arising through the formation and processing of slipped-strand DNAs. These transient intermediates of repeat length mutations are formed by out-of-register mispairing of repeat units on complementary strands. The three-...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi301369b
更新日期:2013-02-05 00:00:00