Maximal enzyme catalytic activity, kinetic

Any detectable effect on the reaction or behavior of a particular system by the interior wall of the container or reaction vessel. Because proteins can form high-affinity complexes with glass and plastic surfaces, one must exercise caution in the choice of reaction kinetic conditions. Wall effects can be discerned if one determines catalytic activity under different conditions that minimize or maximize contact of the solution with the container. In principle, an enzyme-catalyzed reaction should proceed at the same rate if placed in a capillary or a culture tube however, contact with the wall is maximized in a capillary, and wall effects should be more prominent. Some investigators add bovine serum albumin to prevent adsorption of their enzyme onto the container s walls. 

Type II deiodinase activity is low in unsupplemented tissue homogenates but is stimulated by DTT [71-74,82,83] and to a lesser extent also by GSH [72]. The DTT concentrations required for maximal enzyme stimulation in the CNS and pituitary seem higher than in BAT and also than those necessary for the type I deiodinase in liver and kidney. Kinetic analysis of the deiodination of varying substrate (T4, rT3) concentrations at different cofactor (DTT) levels have indicated a sequential reaction mechanism for the type II deiodinase [73,82,83]. This is very suggestive of the formation of a ternary enzyme-substrate-cofactor complex in the catalytic process [82], The physiological cofactor of the type II deiodinase has not been identified but it has been observed that GSH depletion with diamide or diethylmaleate impairs T4 to T3 conversion in GH3 pituitary tumor cells [93]. 

The substrate concentration at which an enzyme reaches one-half of its maximal catalytic activity is often used as a measure of the sensitivity of an enzyme to substrate saturation. This particular substrate concentration usually has about the same numerical value as Km, sometimes known as the Michaelis-Menten constant for the enzyme. The maximum rate of reaction per mole of enzyme is often given the symbol cat. and the maximum rate of reaction for a given enzyme concentration is often symbolized as Vmax- Often, the kinetics of more complex enzyme-catalyzed reactions can be placed in this form under some restricted range of conditions [1]. 

Different from conventional chemical kinetics, the rates in biochemical reactions networks are usually saturable hyperbolic functions. For an increasing substrate concentration, the rate increases only up to a maximal rate Vm, determined by the turnover number fccat = k2 and the total amount of enzyme Ej. The turnover number ca( measures the number of catalytic events per seconds per enzyme, which can be more than 1000 substrate molecules per second for a large number of enzymes. The constant Km is a measure of the affinity of the enzyme for the substrate, and corresponds to the concentration of S at which the reaction rate equals half the maximal rate. For S most active sites are not occupied. For S >> Km, there is an excess of substrate, that is, the active sites of the enzymes are saturated with substrate. The ratio kc.AJ Km is a measure for the efficiency of an enzyme. In the extreme case, almost every collision between substrate and enzyme leads to product formation (low Km, high fccat). In this case the enzyme is limited by diffusion only, with an upper limit of cat /Km 108 — 109M. v 1. The ratio kc.MJKm can be used to test the rapid... 

Affinity labeling agents are intrinsically reactive compounds that initially bind reversibly to the active site of the enzyme then undergo chemical reaction (generally an acylation or alkylation reaction) with a nucleophile on the enzyme (Scheme 8). To differentiate a reversible inhibitor from an irreversible one, often the dissociation constant is written with a capital i, K (65), instead of a small i, K, which is used for reversible inhibitors. The K denotes the concentration of an inactivator that produces half-maximal inactivation. Note that this kinetic Scheme is similar to that for substrate turnover except instead of the catalytic rate constant, kcat for product formation, kmact is used to denote the maximal rate constant for inactivation. 

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