Activities enzymes

The enzyme activity is used as an index to estimate the enzyme s potential to produce the desired product. Different physicochemical techniques are available to measure the activity, either by substrate consumption or product formation (Smeltzer et al, 1992), For a good estimate of the activity, the range of substrate concentrations should be carefully selected and accurately measured. Continuous assays, which continuously monitor changes in the reaction solution s physical properties, such as light absorbance (colorimetric), fluorescence (fluorometric), or heat release/ absorbance (calorimetric), are among the possible techniques. Discontinuous assays, where samples from a reaction solution are collected at intervals, and the amount of substrate or product concentrations are measured, are also frequently used. 

Lipase activity can be measured using the aforementioned methods and are similar to other enzymatic reactions (Beisson et al., 2000). Choosing the appropriate method depends on lipase sensitivity, the availability of substrates, and the ease of measurement (Hendrickson, 1994 Singh and Mukhopadhyay, 2012). 

As discussed above, the most common indicator of enzyme activity is KM, which is determined by measuring Vmas at enzyme saturation conditions and Ve at a concentration of S, [S], near to the saturation concentration of S for a given concentration of E, [E]. V0 is taken as the initial slope of the plot of reaction conversion vs time for that value of [S], and a series of such plots and V values are obtained at different values of [E], The slopes of a plot of Ve vs [E] then gives VC/[E], which can be used to calculate KM from the Michaelis-Menten equation. 

Three other parameters are also used to evaluate enzyme activity these are  

HPLC/MS/MS Quantitative Assays of Cytochrome P450 Enzyme Activity 

FIGURE 12.11 Metabolism of bupropion by CYP2B6 to hydroxy-bupropion and the structure of the stable iso tope-labeled internal standard [ H ]hydroxybupropion. 

The HPLC/MS/MS assays of other CYP enzymes are very similar in principle and use the identical instrumentation but employ different internal standards. As a consequence of the high degree of specificity of MS/MS selected reaction monitoring, batteries of CYP assays can be robotically programmed for high throughput with little additional manpower. 

HPLC/UV and Immunoassays of Cyclosporine Assays for Therapeutic Drug Monitoring 

FIGURE 12.12 Electrospray ionization mass spectra of bupropion (solid line) and [ Hgjhydroxy-bupropion (dotted line). Note that the protonated molecular ions (MH+, respectively, at mjz 256 and 262) exhibit characteristic chlorine isotope peaks that have 25% of the molecular ion intensity at m/z 258 and 264, due to the relative natural abundance of Cl and Cl. This is reflected also in the MH+-H2O ions at mjz 238 and 244. Data provided by R.L. Walsky and R.S. Obach, Pfizer, New York, NY. 

The activities of several enzymes have been studied in partially hydrated powders as a funcuon of water activity or water content. Experiments of this type are not difficult to perform. Solutions of substrate and enzyme are mixed quickly, and the mixture is immediately frozen and lyophilized, which stops the reaction and gives a stable dry powder. If appropriately high concentrations of enzyme and substrate are mixed, the powder is of the enzyme-substrate complex. The sample is rehydrated under a controlled atmosphere to give the desired final hydration level. Conditions, particularly the pH of the sample, are set such that the hydration equilibrium is substantially complete (within several hours) before appreciable enzyme reaction has taken place. The problem of defining pH in partially hydrated powders was discussed in Section II,D in connection with hydrogen-exchange measurements. The pH of a powder appears to equal the nominal pH (that of the solution from which the powder was lyophilized) above about 0.15 A. 

In solution the hexasaccharide is cleaved by lysozyme relatively cleanly to tetramer and dimer. This is true also in the hydrated powder, at hydrations below 40 wt% water. Between 40 wt% water and the dilute solution the pattern undergoes changes, reflecting the contribution of transfer reactions. The reaction rate at full hydration in the powder (i.e., 0.38 h) is about 10% of the solution rate. 

Skujins and McLaren (1967) co-lyophilized urease and [ CJurea. The rate of reaction, determined by the level of C02, was measured as a function of water content. Onset of enzyme reaction occurred at 0.6 relative humidity. The samples contained a 25 1 weight ratio of urea to urease. Sorption isotherms measured separately for enzyme and urea showed that below 0.75 relative humidity the urea adsorbed no water, and thus that the enzyme changes reflected adsorption of water by the urease. From the sorption isotherm for urease, 0.6 relative humidity corresponds to 0.15 h. 

One enzyme reaction has been detected at extremely low hydration. Yagi et al. (1969) found that hydrogenase lyophilized at 10 mm pressure catalyzed the para-hydrogen-ortho-hydrogen conversion. 

Stevens and Stevens (1979) measured the hydration dependence of glucose-6-phosphate dehydrogenase, hexokinase, fumarate hydratase (fumarase), and glucose-6-phosphate isomerase (phosphoglucose isom-erase) over the range 0.1-0.6 h. Serum albumin was used as a carrier protein to buffer the water content. The hydration isotherms of the enzymes and the serum albumin were assumed to be similar. For the first three enzymes activity was detected (0.05% of full solution activity) near 0.2 h. Activity was measurable for the isomerase at 0.15 h. In all cases, even at 0.3 h, the activity in the powder was less than 5% of the solution rate. Diffusion of substrates in the powder may be rate limiting. The amount of albumin in the powder affected the rate. 

The use of appropriate normal ranges is important in evaluating abnormal levels of plasma enzymes. However, an abnormal isoenzyme pattern may occur despite normal total activity (see above). The standard unit for enzyme activity was discussed in Chapter 6. The normal range is affected by a variety of factors age, sex, race, degree of obesity, pregnancy, alcohol or other drug consumption. 

Enzyme activities may also be measured in urine, cerebrospinal fluid, bone marrow cells or fluid, amniotic cells or fluid, red blood cells, leukocytes, and tissue cells. Cytochemical localization is possible in leukocytes and biopsy specimens (e.g., from liver and muscle). Under ideal conditions, both the concentration of the enzyme and its activity would be measured. Radioimmunoassay (RIA) and its alternative modes such as fluorescence immunoassay (FIA), fluorescence polarization immunoassay (FPIA), and chemiluminescence immunoassay (CLIA) (discussed later), can be used to measure enzyme concentration as well as other clinically important parameters. 

Diagrammatic representation of the measurement of the activity of an enzyme, showing product formation as a function of time. The true activity of the enzyme is calculated from data obtained when the reaction rate is linear with maximum velocity. The reaction rate is directly proportional to the amount of active enzyme only when the substrate concentrations are maintained at saturating levels and when other variables (e.g., pH, and cofactors) are held constant at optimal conditions. 

Red cell glucose-6-phosphate dehydrogenase (Chapter 15) can be specifically assayed in a red cell hemolysate. The enzyme catalyzes the reaction 

Absorption spectra of NAD+ (NADP ) and NADH (NADPH). At 340 nm, the reduced coenzymes (NADH or NADPH) show significant absorbance, whereas the oxidized forms (NAD or NADP ) show negligible absorbance. Thus, many enzymatic reactions can be monitored at 340 nm if the reaction is directly or indirectly dependent upon a dehydrogenase reaction involving a nicotinamide adenine dinucleotide as a coenzyme. 

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Radmacher M, Fritz M, Hansma H G and Hansma P K 1994 Direct observation of enzyme activity with the atomic force microscope Science 265 1577... 

All organisms seem to have an absolute need for magnesium. In plants, the magnesium complex chlorophyll is the prime agent in photosynthesis. In animals, magnesium functions as an enzyme activator the enzyme which catalyses the ATP hydrolysis mentioned above is an important example. 

Figure 10.3-23. Metabolic model of glycolysis and tbe pentose phosphate pathway in E. coli. Squares Indicate enzyme activities circles indicate regulatory effects,...

This enzyme, sometimes also called the Schardinger enzyme, occurs in milk. It is capable of " oxidising" acetaldehyde to acetic acid, and also the purine bases xanthine and hypoxanthine to uric acid. The former reaction is not a simple direct oxidation and is assumed to take place as follows. The enzyme activates the hydrated form of the aldehyde so that it readily parts w ith two hydrogen atoms in the presence of a suitable hydrogen acceptor such as methylene-blue the latter being reduced to the colourless leuco-compound. The oxidation of certain substrates will not take place in the absence of such a hydrogen acceptor. 

Beyond pharmaceutical screening activity developed on aminothiazoles derivatives, some studies at the molecular level were performed. Thus 2-aminothiazole was shown to inhibit thiamine biosynthesis (941). Nrridazole (419) affects iron metabohsm (850). The dehydrase for 5-aminolevulinic acid of mouse liver is inhibited by 2-amino-4-(iS-hydroxy-ethyl)thiazole (420) (942) (Scheme 239). l-Phenyl-3-(2-thiazolyl)thiourea (421) is a dopamine fS-hydroxylase inhibitor (943). Compound 422 inhibits the enzyme activity of 3, 5 -nucleotide phosphodiesterase (944). The oxalate salt of 423, an analog of levamisole 424 (945) (Scheme 240),... 

Very Htfle data are available regarding effects of anaboHc steroid implants on the Hpid metaboHsm in growing mminants. Lipogenic enzyme activity and fatty acid synthesis in vitro were elevated in subcutaneous adipose tissue from bulls implanted with estradiol (44), which may account for the increase in fat content of carcasses reported in some studies. TBA implants have no effect on Hpogenesis in intact heifers, and only tend to reduce Hpogenic enzyme activities in ovariectomized heifers (45). 

In fact, most RIAs and many nonisotopic immunoassays use a competitive binding format (see Fig. 2). In this approach, the analyte in the sample to be measured competes with a known amount of added analyte that has been labeled with an indicator that binds to the immobilized antibody. After reaction, the free analyte—analyte-indicator solution is washed away from the soHd phase. The analyte-indicator on the soHd phase or remaining in the wash solution is then used to quantify the amount of analyte present in the sample as measured against a control assay using only an analyte-indicator. This is done by quantifying the analyte-indicator using the method appropriate for the assay, for example, enzyme activity, fluorescence, radioactivity, etc. 

The specific enzyme to be used in an EIA is deterrnined according to a number of parameters including enzyme activity and stabiUty (before, during, and after conjugation), cost and availabiUty of the enzyme substrate, and the desired end point of the EIA, such as color. Most EIAs utilize a colored end point which can be readily deterrnined both visually and spectrophotometricaHy. Table 1 Hsts a number of enzymes which have been used in immunoassays and their substrates. 

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 biomedical and biological appHcations of mictocapsules continue to be developed. For example, the encapsulation of enzymes continues to attract interest even though loss of enzyme activity due to harshness of the encapsulation protocols used has been a persistent problem (59). The use of mictocapsules in antibody hormone immunoassays has been reviewed (60). The encapsulation of hemoglobin as a ted blood substitute has received much attention because of AIDS and blood transfusions (61). 

Potassium is required for enzyme activity in a few special cases, the most widely studied example of which is the enzyme pymvate kinase. In plants it is required for protein and starch synthesis. Potassium is also involved in water and nutrient transport within and into the plant, and has a role in photosynthesis. Although sodium and potassium are similar in their inorganic chemical behavior, these ions are different in their physiological activities. In fact, their functions are often mutually antagonistic. For example, increases both the respiration rate in muscle tissue and the rate of protein synthesis, whereas inhibits both processes (42). 

Specificity for a particular charged substrate can be engineered into an enzyme by replacement of residues within the enzyme-active site to achieve electrostatic complementarity between the enzyme and substrate (75). Protein engineering, when coupled with detailed stmctural information, is a powerful technique that can be used to alter the catalytic activity of an enzyme in a predictable fashion. 

Chemical Pathology. Also referred to as clinical chemistry, this monitoring procedure involves the measurement of the concentration of certain materials in the blood, or of certain enzyme activities in semm or plasma. A variety of methods exist that allow (to variable degrees of specificity) the definition of a particular organ or tissue injury, the nature of the injurious process, and the severity of the effect (76). 

Over 250 analogues of the B vitamers have been reported (11,100). Nearly all have low vitamin B activity and some show antagonism. Among these are the 4-deshydroxy analogue, pyridoxine 4-ethers, and 4-amino-5-hydroxymeth5i-2-methyipyrimidine, a biosynthetic precursor to thiamine. StmcturaHy unrelated antagonists include dmgs such as isoniazid, cycloserine, and penicillamine, which are known to bind to pyridoxal enzyme active sites (4). 

Thiamine requirements vary and, with a lack of significant storage capabiHty, a constant intake is needed or deficiency can occur relatively quickly. Human recommended daily allowances (RDAs) in the United States ate based on calorie intake at the level of 0.50 mg/4184 kj (1000 kcal) for healthy individuals (Table 2). As Httle as 0.15—0.20 mg/4184 kJ will prevent deficiency signs but 0.35—0.40 mg/4184 kJ are requited to maintain near normal urinary excretion levels and associated enzyme activities. Pregnant and lactating women requite higher levels of supplementation. Other countries have set different recommended levels (1,37,38). 

Enzyme Assays. An enzyme assay determines the amount of enzyme present in sample. However, enzymes are usually not measured on a stoichiometric basis. Enzyme activity is usually determined from a rate assay and expressed in activity units. As mentioned above, a change in temperature, pH, and/or substrate concentration affects the reaction velocity. These parameters must therefore be carefully controlled in order to achieve reproducible results. 

Potentiometry is another useful method for determining enzyme activity in cases where the reaction Hberates or consumes protons. This is the so-called pH-stat method. pH is kept constant by countertitration, and the amount of acid or base requited is measured. An example of the use of this method is the determination of Hpase activity. The enzyme hydroly2es triglycerides and the fatty acids formed are neutralized with NaOH. The rate of consumption of NaOH is a measure of the catalytic activity. 

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