Enzymes activity, kinetic measurements

Regardless of the assay used, the non-hnearity of cellulase kinetics requires that the enzyme activity be measured based on a fixed level of conversion. 

One of the most valuable applications of electronic data-processing is in enzyme assays. Kinetic measurements of enzyme activity in which the rate of reaction is monitored (usually using UV measurement) are more specific than endpoint colorimetric methods, in which the development of color in a coupled reaction is measured after a fixed time. Modern systems continuously or intermittently monitor the growth in concentration of the reaction product or the decrease in concentration of one of the reactants (the substrate). From the rate of change in concentration or the average change in concentration over several fixed time-intervals, the circuitry calculates the activity in reportable units. Other ramifications of these systems will be discussed below. 

The IMI herbicides also exhibit complex interactions with AHAS. When enzyme activity was measured over an extended period in the presence of various concentrations of imazapyr, inhibition increased with time, thereby suggesting that the equilibrium between the herbicide and AHAS was reached slowly, a characteristic of tight-binding inhibitors [51]. In contrast to SUs, substrate-inhibitor studies suggested that inhibition by imazapyr is uncompetitive with respect to pyruvate, which implies that the synthetic molecule binds to AHAS only after formation of the ternary enzyme-pyruvate-ThDP complex [52]. However, noncompetitive binding has also been reported for the IMIs, which underscores the complexity of the kinetics of AHAS inhibition [49]. 

Escherichia coli RNA-polymerase hole- and core enzymes were 95% pure by sodium dodecyl sulfate (SDS) gel electrophoresis. Enzyme activity was measured as the amount of [ C]AMP or [ C]UMP incorporated into acid-insoluble material after a 10-min incubation at 37°. The assay mixture contained in 0.1 ml 40 mM Tris-chloride at pH 8.0, 8 mM MgCl2, 5 mAf dithioerythritol, 0.2 Azoo unit of poly[d(AT)], 0.05 M KCl, 1 mAf ATP, and 1 mAf [ C]UTP. For kinetic studies, a fixed concentration of ATP (0.4 mAf) was used and the concentration of [ C]UTP was varied. Enzyme activity was measured as the amount of [ C]UTP incorporated into acid-insoluble material after 5 min. 

Eor measurement of a substrate by a kinetic method, the substrate concentration should be rate-limiting and should not be much higher than the enzyme s K. On the other hand, when measuring enzyme activity, the enzyme concentration should be rate-limiting, and consequentiy high substrate concentrations are used (see Catalysis). 

Other analyzers such as the Gilford Automated Enzyme Analyzer and the LKB-8600 Reaction Rate Analyzer analyze discrete samples one at a time. These instruments provide kinetic analyses, digital data reduction at the time each sample is analyzed, and excellent electronic and optical characteristics. Recently, Atwood has developed kinetic enzyme analyzers which require only 9 seconds for measuring an enzyme activity, using highly stable and sensitive electronic circuits (12). This short read out time allows a large number of samples to be processed by one instrument in an automated mode. 

In addition to enzyme activity, the concentration of an nonelectroactive substrate can be determined electrochemically by this technique. By keeping the substrate (analyte) the limiting reagent, the amount of product produced is directly related to the initial concentration of substrate. Either kinetic or equilibrium measurements can be used. Typically an enzyme which produces NADH is used because NADH is readily detected electrochemically. Lactate has been detected using lactate dehydrogenase, and ethanol and methanol detected using alcohol dehydrogenase... 

Capacitance measurements of phospholipid monolayers at the ITIES have been proposed as a suitable tool for studying the enzyme activity under the precise control of the electrical state of the monolayer [81]. Kinetics of hydrolysis of phosphatidylcholine... 

It was suggested,1 on the basis of kinetic measurements, that the phosphorofluoridates inhibit esterases by virtue of a highly specific affinity for the active centres of this group of enzymes. Preliminary experiments by Boursnell and Webb2 with diisopropyl phosphorofluoridate containing 32P gave results which were in accordance with this view. 

Other automated systems may be purchased for a specific purpose and are called dedicated instruments, e.g. glucose analyser. Others have fairly restricted applications, an example being the reaction rate analysers which are specifically designed for the kinetic measurements of enzyme activity. Some of the more recently developed instruments employ individual pre-prepared disposable test packs or strip devices which contain all the reagents for each particular assay in a dry form. 

Removal of calcium from HRP C has a significant effect not only on enzyme activity and thermal stability, but also on the environment of the heme group. The calcium-depleted enzyme has optical, EPR, and H NMR spectra that are different from those of the native enzyme (211). Temperature dependence studies indicate that the heme iron exists as a thermal admixture of high- and low-spin states. Kinetic measurements at pH 7 show that ki, the rate constant for compound I formation, is only reduced marginally from 1.6 0.1 x 10 to 1.4 x lO M s , whereas k, the rate constant for compound II reduction, is reduced from 8.1 1.6 x 10 to 3.6 x lO M s (reducing substrate p-aminobenzoic acid), 44% of its initial value (211). There can be little doubt that this is the main reason for the loss of enzyme activity on calcium removal. 

In most kinetic investigations, one assumes the enzyme remains stable over the course of the measurement. When this is the case, corrective measures must be taken to obtain valid kinetic data. A useful test for any enzyme system is to plot enzyme activity versus time. This is readily accomplished by using a standardized assay (usually at optimal or saturating substrate concentrations) to measure the enzyme s specific activity periodically during the course of some experiment. This approach may fail to detect a reduction in activity characterized by lower affinity for substrate however, use of a subsaturating substrate concentration in a time-course study will reveal this behavior. 

In determining enzyme activities, it is usually assumed that at a fixed set of so-called saturating substrate concentrations a sufficiently accurate value of F, ax is obtained. Bisubstrate kinetic analyses of UDP-glucu-ronyltransferase [assayed with bilirubin (P5) and p-nitrophenol (V6), respectively] indicate that a true measure of the amount of enzyme can be obtained only by suitable extrapolation procedures. This restriction applies in particular to bilirubin (A2, HIO, T8) and other aglycons (M15, V6) because of substrate inhibition. UDP-glucuronic acid was inhibitory at concentrations only about 10-fold higher than the apparent Km value (HIO) this was most pronounced at relatively short incubation times. Mg was noninhibitory at concentrations equal to 20 times the apparent Km values (F3, HIO). 

The activity of a very weak active mutant measured by steady state kinetics could result from traces of a wild-type or more active mutant in the preparation either as a contaminant or because of natural errors of misincorporation. The error rate in protein biosynthesis can be as high as one part in 100 or one part in 1000.10 The presence of a small amount of wild-type enzyme in an inactive mutant would give a low value of kcat (which is directly proportional to the concentration of wild type) but the KM value for the wild-type enzyme. Thus, the finding of a low value of kcat and the wild-type KM for a mutant is very suspicious. 

The conditions used in an enzyme assay depend on what is to be accomplished by the assay. There are two primary applications of an enzyme assay procedure. First, it may be used to measure the concentration of active enzyme in a preparation. In this circumstance, the measured rate of the enzyme-catalyzed reaction must be proportional to the concentration of enzyme stated in more kinetic terms, there must be a linear relationship between initial rate and enzyme concentration (the reaction is first order in enzyme concentration). To achieve this, certain conditions must be met (1) the concentrations of substrate(s), cofactors, and other requirements must be in excess (2) the reaction mixture must not contain inhibitors of the enzyme and (3) all environmental factors such as pH, temperature, and ionic strength should be controlled. Under these conditions, a plot of enzyme activity (p-rnole product formed/minute) vs. enzyme concentration is a straight line and can be used to estimate the concentration of active enzyme in solution. 

Before an immobilized enzyme can be used for an industrial process, it is essential to characterize it in terms of its catalytic and kinetic properties. A quantitative assay must be developed to measure the activity, kinetic parameters, and stability of the enzyme. In a coupling reaction, H202 rapidly reacts with phenol and 4-aminoantipyrine (electron donor) in the presence of peroxidase to produce a quinoneimine chromogen (Equation E12.2, Figure El 1.2), which is intensely colored with a maximum absorbance at 510 nm. (This is the same as the product formed in the analysis of cholesterol in Experiment 11.)... 

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