Auxiliary enzyme

In carrying out an enzyme assay it may be convenient to introduce an auxiliary enzyme to the system to effect the removal of a product produced by the first enzymatic reaction. McClure [Biochemistry, 8 (2782), 1969] has described the kinetics of certain of these coupled enzyme assays. The simplest coupled enzyme assay system may be represented as... 

As mentioned above, in order to extend the potentialities of the luminescence-based optical fibre biosensors to other analytes, auxiliary enzymes can be used. The classical approaches consist either of the coimmobilization of all the necessary enzymes on the same membrane or of the use of microreactors including immobilized auxiliary enzymes and... 

Another approach, developed in our laboratory, consists of the compartmentalization of the sensing layer25"27. This concept, only applicable for multi-enzyme based sensors, consist in immobilizing the luminescence enzymes and the auxiliary enzymes on different membranes and then in stacking these membranes at the sensing tip of the optical fibre sensor. This configuration results in an enhancement of the sensor response, compared with the case where all the enzymes are co-immobilized on the same membrane. This was due to an hyperconcentration of the common intermediate, i.e. the final product of the auxiliary enzymatic system, which is also the substrate of the luminescence reaction, in the microcompartment existing between the two stacked membranes. 

An auxiliary enzyme is the enzyme or enzymes that catalyse the intermediary reactions in a coupled enzyme assay. 

Many assays have been described in which the initial product forms the substrate of an intermediary reaction involving auxiliary enzymes. The assay of creatine kinase (EC 2.13.2), for example, involves hexokinase (EC 2.7.1.1) as the auxiliary enzyme and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) as the indicator enzyme ... 

O2). One of the two oxygen atoms is transferred to the substrate, while the other is released as a water molecule. The necessary reducing equivalents are transferred to the actual monooxygenase by an FAD-containing auxiliary enzyme from the coenzyme NADPH+HT... 

Dihydrofolate reductase acts as an auxiliary enzyme for thymidylate synthase. It is involved in the regeneration of the coenzyme N, N -methylene-THF, initially reducing DHF to THF with NADPH as the reductant (see p. 418). The folic acid analogue methotrexate, a frequently used cytostatic agent, is an extremely effective competitive inhibitor of dihydrofolate reductase. It leads to the depletion of N, N -methylene-THF in the cells and thus to cessation of DNA synthesis. 

A protocol for continuous enzyme assay that involves one or more auxiliary enzymes to convert a product of the primary reaction in a second or auxiliary reaction that produces a change in absorbance or fluorescence. As noted below, coupled enzyme assays, while convenient, are fraught with experimental limitations that must be overcome in order to obtain valid initial velocity data. 

The components of the coupling system should neither inhibit nor activate the primary enzyme. Moreover, care must be exercized to ascertain that the auxiliary enzyme (s) is not contaminated with other minor enzyme activities capable of influencing the primary enzymatic activity. The results from any coupled enzyme assay must exactly match the results obtained with other valid initial rate assays to ensure that the presence of the auxiliary system in no way affects the activity of the primary enzyme. This is typically accomplished by comparing data obtained from the coupled assay with stopped-time assay results to ensure that similar results are obtained. 

Auxiliary Enzyme Assay Methods for Initial Rate Analysis ... 

Occasionally, one may also wish to use an auxiliary enzyme not as an assay system but strictly as a means for maintaining the steady-state concentration of a primary reactant in a multisubstrate reaction system. For instance, acetate kinase (and its substrate acetyl phosphate) or creatine kinase (and its substrate creatine phos-... 

The assay protocol should measure true initial rates (See Initial Rate Condition). For most systems, this represents a time period in which less than ten percent of the substrate concentration has undergone conversion. However, if a reaction is not significantly favored thermodynamically or if product inhibition is particularly potent, then a much smaller percentage of substrate conversion may be needed such that true initial rate conditions are obtained. Addition of an auxiliary enzyme system may prove necessary to avoid product accumulation. See Coupled Enzyme Assays... 

The initial-rate phase may quickly end if one or more products accumulate to concentrations approaching the respective inhibition constants. In these cases, one may seek to minimize this problem by including an auxiliary enzyme (a) to remove product(s) or (b) to regenerate the substrate concentration. Ideally, the auxiliary enzyme should have a lower value for the product of the primary reaction than the corresponding value for the primary enzyme. See Chemical Kinetics ATP/GTP regeneration... 

Provide a reference or direct experimental proof that the conditions chosen do provide initial rate measurements. At a minimum, the percentage of substrate consumed during the course of an initial rate determination should be specified. One should also show that under initial rate conditions a doubling of enzyme concentration should exactly produce a doubling in the observed initial rate. Likewise, if an auxiliary enzyme assay is used to monitor the primary enzyme s activity the observed rate at low or high substrate concentration should not depend on the concentration of additional enzyme(s), substrate(s), or factors used for the coupled assay. 2. Describe all assay conditions (eg., concentrations of substrates, products, inhibitors, and/or activators enzyme concentration temperature pH and buffer composition ... 

AUTONOMOUS CATALYTIC DOMAIN AUTOPHAGY AUTOPHOSPHORYLATION AUTOPROTOLYSIS AUTOPROTOLYSIS CONSTANT Auxiliary enzyme assays,... 

Many of the fairly large number of enzyme thermistor biosensors reported so far have been used for the determination of biological substrates or, to a much lesser extent, inorganic substrates. Experimental set-ups similar to that depicted in Fig. 3.22.C were used to determine the substrates listed in Table 3.3, which also gives the primary enzymes and any auxiliary enzymes or reagents employed to improve the determination [158]. 

Analyte Main enzyme Auxiliary enzyme or reagent (effect)... 

As depicted in Figure 6.8 the stability screening was based on DERA activity assay, the retro-aldol reaction of 2-deoxy-D-ribose 5-phosphate to acetaldehyde and D-glyceraldehyde 3-phosphate. D-glyceraldehyde 3-phosphate is further converted by the auxiliary enzymes triose phosphate isomerase and glycerol phosphate dehydrogenase. As the latter reaction consumes NADH it can be measured spectro-pho to metrically by the decrease in absorbance at 340 nm. 

The other auxiliary enzyme (a reductase) is required for oxidation of polyunsaturated fatty acids—for... 

A second important difference between mitochondrial and peroxisomal fi oxidation in mammals is in the specificity for fatty acyl-CoAs the peroxisomal system is much more active on very-long-chain fatty acids such as hexacosanoic acid (26 0) and on branched-chain fatty acids such as phytanic acid and pristanic acid (see Fig. 17-17). These less-common fatty acids are obtained in the diet from dairy products, the fat of ruminant animals, meat, and fish. Their catabolism in the peroxisome involves several auxiliary enzymes unique to this organelle. The inability to oxidize these compounds is responsible for several serious human diseases. Individuals with Zellweger syndrome are unable to make peroxisomes and therefore lack all the metabolism unique to that organelle. In X-linked adrenoleukodystrophy (XALD), peroxisomes fail to... 

An understanding of protein synthesis, the most complex biosynthetic process, has been one of the greatest challenges in biochemistry. Eukaryotic protein synthesis involves more than 70 different ribosomal proteins 20 or more enzymes to activate the amino acid precursors a dozen or more auxiliary enzymes and other protein factors for the initiation, elongation, and termination of polypeptides perhaps 100 additional enzymes for the final processing of different proteins and 40 or more kinds of transfer and ribosomal RNAs. Overall, almost 300 different macromolecules cooperate to synthesize polypeptides. Many of these macromolecules are organized into the complex three-dimensional structure of the ribosome. 

Unsaturated fatty acids. Mitochondrial P oxidation of such unsaturated acids as the A9-oleic acid begins with removal of two molecules of acetyl-CoA to form a A5-acyl-CoA. However, further metabolism is slow. Two pathways have been identified (Eq. 17-l).26 29b The first step for both is a normal dehydrogenation to a 2-fraus-5-czs-dienoyl-CoA. In pathway I this intermediate reacts slowly by the normal p oxidation sequence to form a 3-czs-enoyl-CoA intermediate which must then be acted upon by an auxiliary enzyme, a ds-AMra s-A2-enoyl-CoA isomerase (Eq. 17-1, step c), before P oxidation can continue. 

In this section we have seen that fatty acids are oxidized in units of two carbon atoms. The immediate end products of this oxidation are FADH2 and NADH, which supply energy through the respiratory chain, and acetyl-CoA, which has multiple possible uses in addition to the generation of energy via the tricarboxylic acid cycle and respiratory chain. Unsaturated fatty acids can also be oxidized in the mitochondria with the help of auxiliary enzymes. Ketone body synthesis from acetyl-CoA is an important liver function for transfer of energy to other tissues, especially brain, when glucose levels are decreased as in diabetes or starvation. 

Glyoxylate cycle. A pathway that uses acetyl-CoA and two auxiliary enzymes to convert acetate into succinate and carbohydrates. 

After one further -oxidation cycle, a 4-cis-enoyl CoA intermediate is formed. It is acted upon by enoyl-CoA dehydrogenase to give 2-trans, 4-cis-dienoyl CoA. Further metabolism of this intermediate proceeds through one cycle of /3-oxidation and requires a second auxiliary enzyme, 2,4-dienoyl-CoA reductase which has high activity in mitochondria. Thus, nine molecules of acetyl-CoA are produced from the oxidation of linoleic acid. 

The initial concentration of the solution was 10.0 g of ( )-(62) in 50 g of acetone. In all runs, 10 mg of seed crystals were used. From the 10 runs highlighted in the 18.1, 21.0 g of /2-(-)-(62) of >92.0% ee and 21.4 g of (jS)-(+)-(62) of >90% ee are obtained from an input of 50.4 g of racemate. The table also nicely illustrates the continuous nature of the process, which coupled with the fact that no resolving agent, chiral auxiliary, enzyme, or catalyst is needed, underlines the economic advantages of this type of process. 

Fig. 3.1. A, The respiratory chain. Q and c stand for ubiquinone and cytochrome c, respectively. Auxiliary enzymes that reduce ubiquinone include succinate dehydrogenase (Complex II), a-glycerophosphate dehydrogenase and the electron-transferring flavoprotein (ETF) of fatty acid oxidation. Auxiliary enzymes that reduce cytochrome c include sulphite oxidase. B, Thermodynamic view of the respiratory chain in the resting state (State 4). Approximate values are calculated according to the Nernst equation using oxidoreduction states from work by Muraoka and Slater, (NAD, Q, cytochromes c c, and a oxidation of succinate [6]), and Wilson and Erecinska (b-562 and b-566 [7]). The NAD, Q, cytochrome b-562 and oxygen/water couples are assumed to equilibrate protonically with the M phase at pH 8 [7,8]. E j (A ,/ApH) for NAD, Q, 6-562, and oxygen/water are taken as —320 mV ( — 30 mV/pH), 66 mV (- 60 mV/pH), 40 mV (- 60 mV/pH), and 800 mV (- 60 mV/pH) [7-10]. FMN and the FeS centres of Complex I (except N-2) are assumed to be in redox equilibrium with the NAD/NADH couple, FeS(N-2) with ubiquinone [11], and cytochrome c, and the Rieske FeS centre with cytochrome c [10]. The position of cytochrome a in the figure stems from its redox state [6] and its apparent effective E -, 285 mV in...

Frequently, the product of a reaction cannot be detected and quantitated direcdy but it is possible to add an auxiliary enzyme that converts the product quantitatively to another substance that can be measured. The overall reaction sequence is ... 

If the auxiliary enzyme is very expensive, then we may wish to calculate the minimum amount needed to complete the reaction in a reasonable time by solving for... 

Pyruvate formed in the ALT reaction is reduced to lactate by LD. The substrate, NADH, and the auxiliary enzymes, MD or LD, must be present in sufficient quantity so that the reaction rate is limited only by the amounts of AST and ALT, respectively As the reactions proceed, NADH is oxidized to NAD. The disappearance of NADH is followed by measuring the decrease in absorbance at 340 nm for several minutes, either continuously or at frequent intervals. The change in absorbance per minute (AA/min) is proportional to the micromoles of NADH oxidized and in turn to micromoles of substrate transformed per minute. A preliminary incubation period is necessary to ensure that NADH-dependent... 

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