Enzyme kinetics

A reaction happens need the energy to vibrate the molecules and the reactant concentration enough. The energy here is often provided by heat. However, in living system, high temperature may harm the biological structure Truly that the concentration in living system is very low. So living organisms solve these problems by using enzyme

94p zingzing09 21-10-2012 66 9   Download

Bài giảng Sinh hoá đại cương (Sinh hoá tĩnh): Chương 4 - Enzymes, cung cấp cho người học những kiến thức như năng lượng; ứng dụng của enzyme; phản ứng hóa học; định nghĩa enzyme; phân loại enzyme; kinetic của enzyme; điều hòa hoạt động enzyme. Mời các bạn cùng tham khảo!

60p khanhchi2590 10-06-2024 4 1   Download

The selective effects of these inhibitors are governed by substituent at position 5. Kinetic studies and molecular docking simulation were performed for elucidating mechanisms of enzyme-inhibitor complex formation.

12p tocectocec 25-05-2020 7 0   Download

The design of b-glycosidases with planed substrate specificity for biotechnological application has received little attention. This is mostly a consequence of the lack of data on the molecular basis of the b-glycosidase specificity, namely data on the energy of the noncovalent interactions in the enzymetransition state complex. In an attempt to fill this gap, sitedirected mutagenesis and enzyme steady-state kinetic experiments with different substrates were conducted, using as model a digestive b-glycosidase (glycoside hydrolase family 1) from Spodoptera frugiperda (Lepidoptera) (Sfbgly50).

10p system191 01-06-2013 44 4   Download

Pyruvate decarboxylase (EC 4.1.1.1) was isolated and purified from the yeast Kluyveromyces lactis. The properties of this enzyme relating to the native oligomeric state, the subunit size, the nucleotide sequence of the coding gene(s), the catalytic activity, and protein fluorescence as well as circular dichroism are very similar to those of the well characterized pyruvate decarboxylase species from yeast.

8p system191 01-06-2013 44 3   Download

Microcalorimetry and UV-vis spectroscopy were used to conduct thermodynamic and kinetic investigations of the scission of calf thymus DNA catalyzed by bleomycin A5 (BLM-A5) in the presence of ferrous ion and oxygen. The molar reaction enthalpy for the cleavage, the Michaelis– Menten constant for calf thymus DNA and the turnover number of BLM-A5 were calculated by a novel thermokinetic method for an enzyme-catalyzed reaction to be )577 ± 19 kJÆmol)1, 20.4 ± 3.8 lM and 2.28 ± 0.49 · 10)2 s)1, respectively, at 37.0 °C. This DNA cleavage was a largely exothermic reaction....

9p system191 01-06-2013 31 4   Download

VanXYC, a bifunctional enzyme from VanC-phenotype Enterococcus gallinarum BM4174 that catalyses D,D-peptidase and D,D-carboxypeptidase activities, was purified as the native protein, as a maltose-binding protein fusion and with an N-terminal tag containing six histidine residues. The kinetic parameters of His6–VanXYC were measured for a variety of precursors of peptidoglycan synthesis involved in resistance: for D-Ala-D-Ala, the Km was 3.6 mM and kcat, 2.5 s)1; for UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-DAla (UDP-MurNAc-pentapeptide[Ala]), Km was 18.8 mM and kcat 6.

7p system191 01-06-2013 40 4   Download

The involvement of the lysine residue present at the active site of Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (Gra3P DH) was investigated by using the lysine specific reagents trinitrobenzenesulfonic acid (TNBS) and pyridoxal phosphate (PP). Both TNBS and PP inactivated EAC cell Gra3P DH with pseudo-first-order kinetics with the rate dependent on modifier concentration. Kinetic analysis, including a Tsou plot, indicated that both TNBS and PP apparently react with one lysine residue per enzyme molecule.

8p system191 01-06-2013 38 3   Download

Tyrosyl-DNA phosphodiesterase (TDP) cleaves the phosphodiester bond linking the active site tyrosine residue of topoisomerase I with the 3¢ terminus of DNA in topoisomerase I–DNA complexes which accumulate during treatment of cancer with camptothecin. In yeast, TDP mutation confers a 1000-fold hypersensitivity to camptothecin in the presence of an additional mutation of RAD9 gene [Pouliot, J.J., Yao, K.C., Robertson, C.A. & Nash, H.A. (1999) Science 286, 552–555].

8p research12 01-06-2013 40 2   Download

Angiotensin converting enzyme (ACE) was already discovered in insects in 1994, but its physiological role is still enigmatic. We have addressed this problem by purifying four new ACE substrates from the ovaries of the grey fleshfly, Neobellieria bullata. Their primary structures were identified as NKLKPSQWISLSD (Neb-ODAIF-11)13), NKLKPSQWI (Neb-ODAIF-11)9), SLKPSNWLTPSE (Neb-ODAIF-2) and LEQIYHL. Database analysis showed significant homology with amino acid sequence stretches as present in the N-terminal part of several fly yolk proteins....

9p research12 01-06-2013 37 3   Download

The prokaryotic ATP-citrate lyase is considered to be a key enzyme of the carbon dioxide-fixing reductive tricarboxylic acid (RTCA) cycle. Kinetic examination of the ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola (Cl-ACL), an a4b4 heteromeric enzyme, revealed that the enzyme displayed typical Michaelis-Menten kinetics toward ATP with an apparent Km value of 0.21 ± 0.04 mM. However, strong negative cooperativity was observed with respect to citrate binding, with a Hill coefficient (nH) of 0.45. ...

8p research12 01-06-2013 43 4   Download

Cloning and over-expression of human glucose 6-phosphate dehydrogenase (Glc6P dehydrogenase) has for the first time allowed a detailed kinetic study of a preparation that is genetically homogeneous and in which all the protein molecules are of identical age. The steady-state kinetics of the recombinant enzyme, studied by fluorimetric initial-rate measurements, gave converging linear Lineweaver–Burk plots as expected for a ternary-complex mechanism.

8p research12 01-06-2013 64 4   Download

A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. ...

11p research12 01-06-2013 43 5   Download

A gene having high sequence homology (45–49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer....

8p research12 01-06-2013 27 4   Download

Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the )1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purification they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower kcat and a slightly higher Km than the wild-type enzyme.

9p research12 01-06-2013 49 4   Download

Arecombinant streptococcalC5apeptidasewas expressed in Escherichia coliand its catalytic properties and thermal sta-bility were subjected to examination. It was shown that the NH2 -terminal region ofC5a peptidase (Asn32–Asp79/ Lys90) forms the pro-sequence segment. Upon maturation the propeptide is hydrolyzed either via an autocatalytic intramolecular cleavage or by exogenous protease strepto-pain. At pH 7.4 the enzyme exhibited maximum activity in the narrow range oftemperatures between 40 and 43
C....

13p research12 29-04-2013 48 3   Download

CTP synthase catalyzes the reaction glutamine + UTP +ATPfiglutamate + CTP + ADP + Pi.The rate of the reaction is greatly enhanced by the allosteric activator GTP.We have studied the glutaminase half-reactionofCTP synthase from Lactococcus lactisand its response to the allosteric activator GTP and nucleotides that bind to the active site. In contrast to what has been found for the Escherichia colienzyme, GTP activation of the L. lactis enzyme did not result in similarkcat values for the glutami-nase activity and glutamine hydrolysis coupled to CTP synthesis. ...

8p research12 29-04-2013 44 2   Download

The present study reports the first purification and kinetic characterization of two plant arogenate dehydrogenases (EC 1.3.1.43), an enzyme that catalyses the oxidative decarboxylation of arogenate into tyrosine in presence of NADP. The twoArabidopsis thalianaarogenate dehy-drogenases TyrAAT1 and TyrAAT2 were overproduced in Escherichia coliand purified to homogeneity. Bio-chemical comparison of the two forms revealed that at low substrate concentration TyrAAT1 is four times more efficient in catalyzing the arogenate dehydrogenase reac-tion than TyrAAT2. ...

9p research12 23-04-2013 48 4   Download

This paper describes violaxanthin de-epoxidation in model lipid bilayers. Unilamellar egg yolk phosphatidylcholine (PtdCho) vesicles supplemented with monogalactosyldi-acylglycerol were found to be a suitable system for studying this reaction. Such a system resembles more the native thylakoid membrane and offers better possibilities for studying kinetics and factors controlling de-epoxidation of violaxanthin than a systemcomposed only of monogalacto-syldiacylglycerol and is commonly used in xanthophyll cycle studies. ...

10p research12 23-04-2013 31 3   Download

Cofactor requirements and enzyme kinetics have been studied of the novel, dual-action enzyme, the isomerizing phycoviolobilinphycoerythrocyanin-a84-cystein-lyase(PVB-PEC-lyase) fromMastigocladus laminosus, which catalyses both the covalent attachment of phycocyanobilin to PecA, the apo-a-subunit of phycoerythrocyanin, and its isomeri-zation to phycoviolobilin.

9p research12 23-04-2013 23 1   Download