Enzymatic reactions progress curve

Although enzyme-catalyzed reactions are described in many other entries in this Handbook, some mention of the time-evolution of an enzymatic process should be considered here. Shown in Fig. 10 is an representation of a typical reaction progress curve. A rapid rise in the concentration of reactant-bound species ES + +... 

Product inhibition is a cause of nonlinearity of reaction progress curves during fixed-time methods of enzyme assay. For example, oxaloacetate produced by the action of aspartate aminotransferase inhibits the enzyme, particularly the mitochondrial isoenzyme. The inhibitory product may be removed as it is formed by a coupled enzymatic reaction malate dehydrogenase converts the oxaloacetate to malate and at the same time oxidizes NADH to NADL... 

Furthermore, in the system with coupled lipase and lipoxygenase, the production rate of HP is governed by the first enzymatic reaction and mass transfer. When TL,- is small (0 to 1 mM equiv. 3 mM LA), the kinetic curve has a sigmoid shape due to surface active properties of LA and HP [25]. Hydrolysis of TL and the increase of LA favor the transfer of LA. Such a transfer allows the lipoxygenase reaction to progress. Since lipox-ygenation consumes LA and produces HP, catalysis and transfer demonstrates a reciprocal influence. 

Figure 6.4 (A) Progress curves for an enzymatic reaction in the presence of increasing concentra-...

Figure 6.14 Progress curves for the enzymatic reaction of dihydrofolate reductase in the presence of the indicated concentrations of methotrexate.

Figure 9-1 The progress curve for an enzymatic reaction in which the substrate S is converted into products.

Fig. 3 (a) Buffer optimization for a pNPP colorimetric assay in 384-well format using p-nitrophenol detection. Activity of HePTP at a concentration of 50 nM was tested in the presence of 1.3 mM pNPP, 1 mM DTT, 0.005 % Tween-20, and various concentrations of Bis-Tris, pH 6.0, and NaCI, over a reaction time of 1 h. The optimal buffer under HTS conditions was 20 mM Bis-Tris, pH. 6.0,1 mM DTT, and 0.005 % Tween-20. The value for pNPP was 0.4 mM. The reaction demonstrated linearity of the progress curves over a period of 2 h. Enzymatic activity was proportional to enzyme concentration. Assay performance was confirmed using the general PTP inhibitor orthovanadate, the ICeo value of which was 150 (b) Buffer optimization for an OMFP... 

The main problem with Eq. (74) is that it accounts only for the velocity of the initial time of the reaction. The information outside the first moments of the inherent progress curve is virtually lost or neglected. Another complication of Eq. (74) is that, even when describing a generalized kinetic, it differs from ordinary chemical curves in its rectangular hyperbola shape instead of the expected exponential form. A further generalized kinetic may instead be assumed, which can be applied to the enzymatic Michaelis-Menten case [196-200] as follows. 

When the reaction is measured spectrophotometrlcally, the progress curve is the net product of the formation the enzyme of compounds absorbing at 390 nm and non-enzymatic reduction of the compounds absorbing at 390 nm. As a result, addition of more substrate after approximately 1050 sec (Fig. 8) resulted in a decrease in absorption. We believe this is due to reduction of the compounds absorbing at 390 nm by pyrocatechol. 

P G. 4. Progress curve of an enzymatic reaction. The velocity of the reaction is the slope of the tangent at the origin of the curve. 

The approach utilized by Burstein and Gut (1969) was to investigate the reaction sequences, outlined above, under conditions of first-order kinetics. An important advantage with first-order reactions is that single points may be used to obtain the individual rates, and detailed progress curves are not required. First-order kinetics with enzymatic reactions have been shown to obtain at relatively low substrate concentrations in which the first-order velocity constants (expressed in time ) are (Dixon and Webb, 1964). Under first-... 

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