Enzymes characterization

Nakayama, N. Matsubara, T. Ohshiro, T., et al., A Novel Enzyme, 2 -Hydroxybiphenyl-2-Sulfinate Desulfinase (DszB), From a Dibenzothiophene-Desulfurizing Bacterium Rhodococcus Erythropolis KA2-5-1 Gene Overexpression and Enzyme Characterization. Biochimica Et Biophysica Acta-Proteins and Proteomics, 2002. 1598(1-2) pp. 122-130. 

Volume 286. Lipases (Part B Enzyme Characterization and Utilization)... 

Kent UM, Mills DE, Rajnarayanan RV, et al. Effect of 17-alpha-ethynylestradiol on activities of cytochrome P450 2B (P450 2B) enzymes characterization of inactivation of P450s 2B1 and 2B6 and identihcation of metabolites. J Pharmacol Exp Ther 2002 300(2) 549-558. 

Using the various simplifications above, we have arrived at a model for reaction 11.9 in which only one step, the chemical conversion occurring at the active site of the enzyme characterized by the rate constant k3, exhibits the kinetic isotope effect Hk3. From Equations 11.29 and 11.30, however, it is apparent that the observed isotope effects, HV and H(V/K), are not directly equal to this kinetic isotope effect, Hk3, which is called the intrinsic kinetic isotope effect. The complexity of the reaction may cause part or all of Hk3 to be masked by an amount depending on the ratios k3/ks and k3/k2. The first ratio, k3/k3, compares the intrinsic rate to the rate of product dissociation, and is called the ratio of catalysis, r(=k3/ks). The second, k3/k2, compares the intrinsic rate to the rate of the substrate dissociation and is called forward commitment to catalysis, Cf(=k3/k2), or in short, commitment. The term partitioning factor is sometimes used in the literature for this ratio of rate constants. 

During purification procedures cellobiase activity was monitored by measuring nitrophenol (at A42onm) release for p-nitrophenyl-/ -D-glucoside (JO). Kinetic studies and enzyme characterization were carried out using / -D-cellobiose as substrate with the product, glucose, measured with a Beckman Glucose Analyzer (JO). Assay conditions were pH 4.8 and 50°C. 

Most enzymes show a bell-shaped pH-velocity profile and a characteristic pH at which their activity is maximal. Figure 5.13 shows V0 vs pH curves for three enzymes. Note that both the pH optimum and the form of the velocity profile vary with the enzyme. Such curves must be interpreted with caution, as they give no indication why the velocity declines above and below the pH optimum. The decline in rate may be due to the formation of improper forms of the enzyme or substrate (or both) or inactivation of the enzyme, or it may be due to a combination of these factors. The possibility of enzyme inactivation is frequently overlooked, although a pH stability curve is necessary for enzyme characterization. A pH stability curve is readily obtained by preincubating the enzyme at a specified pH for a period of time equal to the assay incubation time and then assaying activity at the optimum pH. 

Terada, Y., Fujii, K., Takaha, T., and Kada, S. 1999. Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization Production of cycloamylose. Appl. Environ. Microbiol., 65, 910-915. 

H) Dihydrozeatin formation The double bond of the tZ side chain could be enzymatically reduced by zeatin reductase, an enzyme characterized from immature seeds of P. vulgaris, resulting in DZ.429 Zeatin reductase uses tZ as a substrate, but not cZ, tZR, iP, or tZ O-glucoside, and requires NADPH as a cofactor (Figures 15 and 18).429 The responsible genes for this reaction have not yet been identified. 

The third research project presented by Rohlfing looked at the intrinsic motions of proteins as they influence catalysis and enzymes. Characterizing the intrinsic motions of enzymes is necessary to fully understand how they work as catalysts. As powerful as structure-function relationships are, the motion of these proteins is intimately connected with their catalytic activity and cannot be viewed as static structures. This realization, asserted Rohlfing, could revolutionize and accelerate approaches to biocatalyst design or directed evolution, and could alter understanding of the relations between protein structure and catalytic function. 

A similar enzyme, characterized definitely as an a-rhamnosidase, was purified by Kamiya et al, (190), and a rhamnosidase could be induced in Klebsiella aerogenes (191), A p-nitrophenyl rhamnosidase from Corti-cium rolfsii was only weakly active on naringin (192),... 

Elucidation of the structure of component B (HSHTP) and the discovery of heterodisulfide reductase are two recent successes in work on the biochemistry of methanogens [131,174,178,197,423]. The enzyme heterodisulfide reductase (HR) carries out Reaction (22), regenerating CoM and HSHTP [131,174,423]. HR is found in all methanogens examined [55], and has been proposed to be involved in energy conservation. Two approaches have been used to study this enzyme characterization of the purified enzyme, and studies with membranes and everted vesicles. 

The CHE in normal sera is separated by electrophoresis into 7 to 12 bands, the number depending on the experimental technique used. The forms of CHE differ in molecular size and appear to be aggregates of different numbers of the same basic unit. Of more interest are the atypical (genetic) variants of the enzyme, characterized by diminished activity against acetylcholine and other substrates, which are found in the sera of a small fraction of apparently healthy people. 

Steady-state kinetic conditions presumes catalytic amount of the enzyme and a large supply of the substrate, and the enzymatic rate does not change leading to constant increase in absorbance. The slope of the linear fit is the rate of catalysis and can be used directly in kinetic calculations and enzyme characterization. For each substrate concentration, subtract the vanadate-control rate from the original rate to remove background degradation from the calculations. 

Generally, the reactivating efficacy of oximes depends on their reactivity and affinity for OPC-inhibited enzyme. Their reactivity is derived from the nucleic activity of oxime anion that is bound on the pyridinium ring (8). Oximes differ from each other by the position of the oxime group on the pyridinium ring only. The reactivity of all available oximes is almost the same because their basic structure is very similar (8). The affinity of oximes for intact enzyme, characterized by dissociation constant of enzyme-reactivator complex (Kdls), and for nerve agent-inhibited enzyme, characterized by dissociation constant of inhibited enzyme-reactivator complex (Kr), is determined by various physicochemical factors such as steric compatibility, electrostatic effects, hydrophobic interactions and by the shape and the size of the whole molecule as well as functional groups (22). 

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