Kinetic measurements, enzyme

After the operator has selected the desired method menu of the relevant samples and has started the instrument, all subsequent steps are fully automated. Since 1987 it is also possible to effect a direct identification of the sample so that there are no longer any problems in respect of a dialogue with a central EDP system. The samples are taken from the sample vessel by means of disposable single use pipette tips that are used for one sample only and exchanged via a computer-monitored pipetting unit. This method excludes the possibility of a carry-over between samples. In accordance with the preset conditions, the required slides are automatically moved to the sample dosage unit (see Fig. 23). Samples of 11 pi serum or plasma will be sufficient for kinetic measurements (enzymes), 10 pi of sample for all other tests. As soon as application of the sample has been completed, the slide is moved to the appropriate incubation chamber by means of the slide rotor (see Fig. 23). The chemical reactions take place in these chambers. This is followed by measurement either by reflectometer (end point or kinetic) or a potentiometric measurement unit. 

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). 

This equation is fundamental to all aspects of the kinetics of enzyme action. The Michaelis-Menten constant, KM, is defined as the concentration of the substrate at which a given enzyme yields one-half of its maximum velocity. is the maximum velocity, which is the rate approached at infinitely high substrate concentration. The Michaelis-Menten equation is the rate equation for a one-substrate enzyme-catalyzed reaction. It provides the quantitative calculation of enzyme characteristics and the analysis for a specific substrate under defined conditions of pH and temperature. KM is a direct measure of the strength of the binding between the enzyme and the substrate. For example, chymotrypsin has a Ku value of 108 mM when glycyltyrosinylglycine is used as its substrate, while the Km value is 2.5 mM when N-20 benzoyltyrosineamide is used as a substrate... 

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. 

The use of fluorescent substrates or products permits sensitive kinetic measurement of enzyme reactions to be undertaken and although there are relatively few natural fluorescent substrates, analogues can sometimes be used (Table 8.7). Some products can be converted to fluorescent compounds and can be used in fixed time assays. 

Carbon kinetic isotope effects on enzyme-catalyzed decarboxylations are among the most intensively studied enzyme reactions. This is because of the central role that carbon dioxide plays in plant metabolism and also because precise kinetic measurements are relatively easy to obtain since the carbon dioxide liberated in the reaction can be immediately analyzed using isotope ratio mass spectrometry. 

Storer AC, Darlison MG, Cornish-Bowden A. 1975. The nature of experimental error in enzyme kinetic measurements. Biochem J 151 361. 

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. 

Because enzymic reactions are so sensitive to variations in temperature, suitable care must be exercised such that the temperature of the system is constant and under adequate control. Ideally, the temperature-regulated water bath and/or circulator should reduce any variability to within less than 0.1°C. Before initiating any reaction kinetic measurement, all samples should be thermally equilibrated. This process may take several minutes, depending on the nature of the sample container and the volume of the sample. 

Any experimental technique that discloses the time-re-solved behavior of a chemical/physical process. These approaches allow one to surmount the inherent limitations of steady-state and/or equilibrium kinetic measurements in the detection and quantification of species that comprise the internal equilibria of enzymic catalysis. 

Urea in kidney dialysate can be determined by immobilizing urease (via silylation or with glutaraldehyde as binder) on commercially available acid-base cellulose pads the process has to be modified slightly in order not to alter the dye contained in the pads [57]. The stopped-flow technique assures the required sensitivity for the enzymatic reaction, which takes 30-60 s. Synchronization of the peristaltic pumps PI and P2 in the valveless impulse-response flow injection manifold depicted in Fig. 5.19.B by means of a timer enables kinetic measurements [62]. Following a comprehensive study of the effect of hydrodynamic and (bio)chemical variables, the sensor was optimized for monitoring urea in real biological samples. A similar system was used for the determination of penicillin by penicillinase-catalysed hydrolysis. The enzyme was immobilized on acid-base cellulose strips via bovine serum albumin similarly as in enzyme electrodes [63], even though the above-described procedure would have been equally effective. 

In principle, there are two possible ways to measure this effect. First, there is the end-point measurement (steady-state mode), where the difference is calculated between the initial current of the endogenous respiration and the resulting current of the altered respiration, which is influenced by the tested substances. Second, by kinetic measurement the decrease or the acceleration, respectively, of the respiration with time is calculated from the first derivative of the currenttime curve. The first procedure has been most frequently used in microbial sensors. These biosensors with a relatively high concentration of biomass have a longer response time than that of enzyme sensors. Response times of comparable magnitude to those of enzyme sensors are reached only with kinetically controlled sensors. 

Steady state kinetic measurements on an enzyme usually give only two pieces of kinetic data, the KM value, which may or may not be the dissociation constant of the enzyme-substrate complex, and the kcM value, which may be a microscopic rate constant but may also be a combination of the rate constants for several steps. The kineticist does have a few tricks that may be used on occasion to detect intermediates and even measure individual rate constants, but these are not general and depend on mechanistic interpretations. (Some examples of these methods will be discussed in Chapter 7.) In order to measure the rate constants of the individual steps on the reaction pathway and detect transient intermediates, it is necessary to measure the rate of approach to the steady state. It is during the time period in which the steady state is set up that the individual rate constants may be observed. 

The pKa at high pH is caused by a substrate-independent conformational change in the enzyme, a change that may be monitored directly by physical techniques such as optical rotation and fluorescence yield. Kinetic measurements give the same pKa as that found by these methods. 

The hypothetical reaction mechanism shown in Fig. 10 is a variation of Scheme (3), and is consistent with the redox and pH dependence of the EPR-detectable nickel species (65). Hydrogen is known to undergo heterolytic cleavage (81) it is proposed that this is an intramolecular reaction, leading to the formation of a nickel (II) hydride and a protonated base in the enzyme (Step 1 in Fig. 10). The Ni-C species is postulated to be a protonated Ni(I) species. An alternative formulation for this state would be a dihydrogen complex, Ni(III) H2, as suggested by Crabtree (104). Ultimately the exact mechanism can only be determined by kinetic measurements. 

The allosteric properties of FDPases present an interesting subject for future study. In the case of the liver enzyme the substrate shows positive cooperativity in binding, but no evidence for cooperativity in catalytic activity has been obtained. Perhaps this is because of the high affinity of the enzyme for the substrate, which prevents precise kinetic measurement at low substrate concentration. On the other hand, the substrate has been shown to increase the affinity of the enzyme for AMP, the allosteric inhibitor. 

Kinetic analysis was used to characterize enzyme-catalyzed reactions even before enzymes had been isolated in pure form. As a rule, kinetic measurements are made on purified enzymes in vitro. But the properties so determined must be referred back to the situation in vivo to ensure they are physiologically relevant. This is important because the rate of an enzymatic reaction can depend strongly on the concentrations of the substrates and products, and also on temperature, pH, and the concentrations of other molecules that activate or inhibit the enzyme. Kinetic analysis of such effects is indispensable to a comprehensive picture of an enzyme. 

Measurements of reactions that occur in less than a few seconds require special techniques to speed up the mixing of the enzyme and substrate. One way to achieve this is to place solutions containing the enzyme and the substrate in two separate syringes. A pneumatic device then is used to inject the contents of both syringes rapidly into a common chamber that resides in a spectrophotometer for measuring the course of the reaction (fig. 7.5). Such an apparatus is referred to as a stopped-flow device because the flow stops abruptly when the movement of the pneumatic driver is arrested. In this type of apparatus it is possible to make kinetic measurements within about 1 ms after mixing of enzyme and substrate. 

Enzymes that catalyze reactions with two or more 7T) substrates work in a variety of ways. In some cases, the intermolecular reaction occurs when all the substrates are bound in a common enzyme-substrate complex in others, the substrates bind and react one at a time. A frequent application of kinetic measurements is to distinguish between such alternatives. 

BMost enzymes are sensitive to inhibition by specific agents that interfere with the binding of a substrate at the active site or with conversion of the enzyme-substrate complex into products. Two of the major applications of kinetic measurements are in distinguishing between... 

Fluorescence microspectrophotometry typically provides chemical information in three modes spectral characterization, constituent mapping in specimens, and kinetic measurements of enzyme systems or photobleaching. All three approaches assist in defining chemical composition and properties in situ and one or all may be incorporated into modem instruments. Software control of monochrometers allows precise analysis of absoiption and/or fluorescence emission characteristics in foods, and routine detailed spectral analysis of large numbers of food elements (e.g., cells, fibers, fat droplets, protein bodies, crystals, etc.) is accomplished easily. The limit to the number of applications is really only that which is imposed by the imagination - there are quite incredible numbers of reagents which are capable of selective fluorescence tagging of food components, and their application is as diverse as the variety of problems in the research laboratory. 

Further progress in understanding this effect, as well as others, of the physical structure of cellulose on enzymatic degradation may be expected from combining physicochemical and morphological techniques and from kinetic measurements in heterogeneous enzymatic hydrolysis, applying substrates of well-defined physical structure and isolated components of the enzyme systems. 

At the start of optimization of the reaction system, suitable values for pH and temperature have to be chosen as a function of the properties of the reactants and enzymes. Fortunately, most enzyme reactions operate in a narrow band with respect to pH value (7-10) and temperature (30-50 °C). The initial substrate concentration and, in the case of two-substrate reactions, the stoichiometric ratio of the two reactants, have to be selected. The selected enzyme concentration influences both the achievable space-time-yield as well as the selectivity in the case of undesired parallel or consecutive side reactions. In the case of multi-enzyme systems, the optimal activity ratio has to be found. The activity and stability of all the enzymes involved have to be known as a function of the reaction conditions, before the kinetic measurements are made. Enzyme stability is an important aspect of biocatalytic processes and should be expressed preferably as an enzyme unit consumption number, with the dimension unit of activity per mass of product (such as mole, lb, or kg). In multi-enzyme systems the stability of all the enzymes has to be optimized so that an optimal reaction rate and space-time-yield result. 

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