Optimized enzyme assay

Another reason is that xylo-oligosaccharides of defined structure are very important substrates that serve as model compounds for the optimization of hydrolytic processes and in enzymic assays. The enormous development... 

Product Acceptors. Many enzyme assays use acceptors, as for instance 2-ethylaminoethanol and other aminated alcohols iihich act as acceptors for the phosphoryl product of the reaction catalyzed by alkaline phosphatase (25) (Fig. 4). Hydroxylamine can act as an acceptor for the hydroxyacetone produced by eno-lase and semicarbazide can act as an acceptor for the pyruvate produced by LD. It is necessary to optimize the concentration of such an acceptor before using it routinely as often what may be a theoretically desirable acceptor is in practice superfluous. 

Brain ChAT has a KD for choline of approximately 1 mmol/1 and for acetyl coenzyme A (CoA) of approximately 10pmol/l. The activity of the isolated enzyme, assayed in the presence of optimal concentrations of cofactors and substrates, appears far greater than the rate at which choline is converted to ACh in vivo. This suggests that the activity of ChAT is repressed in vivo. Surprisingly, inhibitors of ChAT do not decrease ACh synthesis when used in vivo this may reflect a failure to achieve a sufficient local concentration of inhibitor, but also suggests that this step is not rate-limiting in the synthesis of ACh [18-20]. 

This section describes recent applications of jitPEC methodologies for separation-based enzymatic assays. It covers the most common applications (1) those involving the development and optimization of assays (2) those in which jitPLC is use to evaluate real-time enzyme kinetics and (3) those in which /./PEC is used to determine substrate specificity. 

Reversible, non-competitive inhibition of polymerase is also afforded by a series of N-benzoyl pyrrolidines. Substitution on the benzoyl moiety with a para-trifluoromethyl group is optimal in this series. Bulky, hydrophobic groups at the 2-position of the pyrrolidine ring increase activity, and the 5-position tolerates a wide range of substituents, indicative of a solvent exposed portion of the inhibitor. Compound (+)-38, containing a 2-thienyl moiety at the 5-position, has an IC50 of 190 nM in the enzyme assay while its enantiomer is almost 100-fold less active [83]. 

Obviously, extrapolation procedures are impractical for routine determination of enzyme activities. When substrate saturation-curves conform to rectangular hyperbolas, reasonable concentrations of substrates should equal 10 to 20 times the respective Km values. As outlined above, application of this rule to assays of bilirubin UDP-glycosyltransferase activities is hampered by substrate inhibition and by occasional deviation from Michaelis-Menten kinetics. The best alternative in such cases may be to choose the concentrations at optimal enzyme activity. However, great care should be exercised in interpreting the results. When a bio-... 

Although a-D-mannosidase from mammalian, plant, and molluscan sources is dependent upon zinc for its catalytic activity, the addition of this ion has a marked effect in the enzyme assay only at those pH values where the active, protein-metal complex dissociates appreciably despite the presence of substrate. (Dissociation, which is greater at lower values of pH, is lessened in the presence of substrate.) The presence of zinc ion in the assay (0.1 mM) is thus of particular importance in the case of the limpet enzyme, where the pH of optimal activity is 3.5. Jack-bean and rat-epididymal a-D-mannosidase are both assayed at pH 5, and up to 10% activation may be observed with zinc. 

To determine whether increased rates of 02 uptake can be observed due to the presence of DPO in a latent form, repeat steps 6 to 12 using the optimized enzyme concentration and adding 0.1 ml of 1 % SDS to the enzyme assay mixture. Reduce the volume of water added by 0.1 ml to maintain a total volume of 3 ml. 

Generally, the nicotinamide coenzymes are not covalently bound to the enzyme. They are employed in enzyme assays and preparative applications by adding catalytical but optimized amounts, and they need to be recycled. For an economic process, an efficient regeneration method is a basic requirement. The necessary recycle number depends essentially on the value of the chiral product, generally the method should recycle the coenzyme 100-100,000 times ([42]). 

Having shown that dibutyryl PC is monomeric under the enzyme assay conditions, we found that the phospholipase A2, which acts poorly on PE in mixed micelles, is activated by dibutyryl PC which is itself an even poorer substrate. 31p-NMR spectroscopy was employed to show that only PE is hydrolyzed in mixtures of various compositions of these two phospholipids. The fully activated enzyme hydrolyzes PE at a similar rate to its optimal substrate, PC containing long-chain fatty acid groups. Because dibutyryl PC is not incorporated into the micelles, these results are consistent with a mechanism of direct activation of the enzyme by phosphoryl-choline-containing lipids (either monomeric or micellar) rather than a change in the properties of the interface being responsible for the activation of phospholipase A2. Therefore, two functional sites on the enzyme have to be assumed an activator site and a catalytic site (6). 

Since MurD was the primary target, it was made the rate-limiting step in the coupled enzyme assay and conditions were optimized for that catalytic reaction. The kinetic parameters were determined for MurD. The Km for UMA and d-G1u guided the choice of substrate concentrations for the MurD reaction so the enzyme was optimally efficient [31], The kinetic parameters for the amino acid or dipeptide and tripeptide substrates for MurE and MurF were then determined... 

Twenty percent (v/v) mycelium suspension was used to inoculate 500-mL conical flasks containing 15 g of corncob as carbon source and 22.5 mL of PPMKC medium (pH6.0) as optimized by Damaso et al. (6). After inoculation, the flasks were incubated in a stationary manner at 45°C for 6 d in a laboratory electric incubator. At each sampling time, the culture medium was vacuum filtered using filter paper (Whatman, no. 4, fast-flow rate), and the filtrate was used for further enzyme assays. During the cultivation, two or more flasks were sampled daily. 

An enzyme assay measures the conversion of substrate to product, under conditions of cofactors, pH and temperature at which the enzyme is optimally active. High substrate concentrations are used so that the initial reaction rate is proportional to the enzyme concentration. Either the rate of appearance of product or the rate of disappearance of substrate is measured, often by following the change in absorbance using a spectrophotometer. Reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), which absorb light at 340 nm, are often used to monitor the progress of an enzyme reaction. 

Enzyme assays The amount of enzyme protein present can be determined (assayed) in terms of the catalytic effect it produces, that is the conversion of substrate to product. In order to assay (monitor the activity of) an enzyme, the overall equation of the reaction being catalyzed must be known, and an analytical procedure must be available for determining either the disappearance of substrate or the appearance of product. In addition, one must take into account whether the enzyme requires any cofactors, and the pH and temperature at which the enzyme is optimally active (see Topic C3). For mammalian enzymes, this is usually in the range 25-37°C. Finally, it is essential that the rate of the reaction being assayed is a measure of the enzyme activity present and is not limited by an insufficient supply of substrate. Therefore, very high substrate concentrations are generally required so that the initial reaction rate, which is determined experimentally, is proportional to the enzyme concentration (see Topic C3). 

Such range finding may frequently straddle the optimal enzyme concentration for assay. Thus, a second series of enzyme dilutions is assayed to pinpoint the enzyme concentration over a narrower range. Usually four to five dilutions over a 10- to 20-fold concentration range are adequate. 

Fluorescence assays for biotransformations are an indispensable tool for enzyme engineering and the daily practice of enzyme studies. Fiuorogenic substrates are particularly useful as general probes for enzyme classes that can be used in routine screening and activity checking. However, they cannot replace the authentic substrate in cases where an optimization towards a particular biotransformation is desired. In such cases an indirect fiuorogenic assay or an instrumental assay may be required in order to follow the reaction. A variety of fluorescence assays for enzymes still remain to be discovered and the development of new enzyme assays... 

The most recent application of RPLC to the analysis of enzymes has been reported by Halfpenny and Brown (HI). An assay for purine nucleoside phosphorylase, a key mediator in the purine salvage pathway, has been developed and optimal conditions for the analysis determined. Figure 20 illustrates the simultaneous separation of the substrate, inosine, and products, uric acid and hypoxanthine. In another analysis. Halfpenny and Brown (H2) developed an assay for hypoxanthine-guanine phos-phoribosyltransferase. Deficiency of this enzyme has been associated with Lesch-Nyhan syndrome as well as primary gout. The activity of the enzyme is determined by measurement of the decrease of the substrate, hypoxanthine, and increase in the product, inosine-5 -monophosphoric acid. A major advantage of using HPLC for enzyme assays is that the simultaneous measurement of both substrate and product reduces the error due to interference from competing enzymes. 

The dimerization behavior caused by changes in the assay temperature has two effects on the behavior of the protease. First, increased temperature increases the catalytic rate of the enzyme, resulting in increased initial rates in the assay, particularly when the enzyme is used directly from a concentrated stock. Second, increased temperature pushes the dimenmonomer equilibrium in the direction of monomer, an apparently less active species, decreasing the steady-state rate of the reaction. In the 60-min end point assay, these effects combine to give optimal enzyme activity at approx 25°C, but further temperature optimization may be useful if initial rates or longer incubation times are used. 

When setting up methods of enzyme assay, it is necessary to (1) explore the relationship between reaction velocity and substrate concentration over a wide range, (2) determine K and (3) detect any inhibition at high substrate concentrations. Zero-order kinetics are maintained if the substrate is present in large excess (i.e., concentrations at least 10 and preferably 100 times that of the value of K ,). When [S] = 10 X K V is approximately 91% of the theoretical y,nax. The K , values for the majority of enzymes are of the order of 10 to 10" mol/L therefore substrate concentrations are usually chosen to be in tlie range of 0.001 to O.lOmol/L. On occasion, the optimal concentrations of substrate cannot be used (e.g., when the substrate has limited solubility or when the concentration of a given substrate inhibits the activity of another enzyme needed in a coupled reaction system). 

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