Three-enzyme system reaction

The oxidation reactions of luminol and lucigenin can be used to assay for H Oj. For example, analysis of glucose in biological systems can be achieved using a three-enzyme system of mutarotase, glucose oxidase and horseradish peroxidase by correlation with the amount of HjOj released. Similarly, cholesterol can be measured using cholesterol oxidase. The fact that the rate of luminol oxidation depends on the concentration of the catalyst can be used as a method for determination of Co +, Fe +, Cr + and Mn + and other catalysts.Some examples of the use of luminol, isolumi-nol and their derivatives in immunoassays are shown in Table 3.11. ... 

Our group has recently cloned a truncated Pd2,6ST containing 17-497 amino acid residues as N-hexohistine tagged protein and explored its application in the one-pot three-enzyme system for preparative synthesis of functionalized o2,6-sialosides (25). The tolerance of donor substrate modification by the purified Pd2,6ST was tested using the one-pot three-enzyme system, in which CMP-sialic acid derivatives were generated in situ from sialic acid precursors by the aldolase and NmCSS. An extremely relaxed donor substrate specificity was observed for Pd2,6ST. The preparative-sacle reactions were then carried out at... 

By controlling the pH and reaction time, PmSTl was used in preparative scale (20-150 mg) synthesis of a2,3-linked sialosides using the one-pot three-enzyme system similar to that described above for the Pd2,6ST. When the reaction was set up at the pH 7.0 to 9.0 for 1 to 2 h at 37 C, only a2,3-linked sialoside was formed and no significant sialidase or a2,6SiaT activitiy was observed under these conditions (24). 

Table 10.1 gives values of rate constants, activation energies, and frequency factors for three enzyme-catalyzed reactions. For comparison, the values for other catalysts are included. Note that molecule for molecule, the enzymes are much more effective catalysts than the nonbiological catalysts. In urease and catalase this higher effectiveness is related to a much smaller activation energy, which is true for a number of other enzyme systems. Enzymes evidently exert their action by allowing the process to occur by a much more favorable reaction path. 

The latter method, called the PI-FEP/UM approach, allows accurate primary and secondary kinetic isotope effects to be computed for enzymatic reactions. These methods are illustrated by applications to three enzyme systems, namely, the proton abstraction and reprotonation process catalyzed by alanine race-mase, the enhanced nuclear quantum effects in nitroalkane oxidase catalysis, and the temperature (in)dependence of the wild-type and the M42W/G121V double mutant of dihydrofolate dehydrogenase. These examples show that incorporation of quantum mechanical effects is essential for enzyme kinetics simulations and that the methods discussed in this chapter offer a great opportunity to more accurately model the mechanism and free energies of enzymatic reactions. 

One of the first examples of concurrent oxidation and reduction reactions in a three-enzyme system was reported in 1996 for the biotransformation of morphine to hydromorphone (Scheme 11.2) [6]. Specifically, the NADPH-dependent morphine-6-dehydrogenase (MDH) was coupled to the NADH-dependent morphinone reductase (MR) through the action of a cofactor aspecific (accepting both NADH and NADPH) glutamate dehydrogenase. The latter cofactor regeneration reaction not only allows the use of catalytic amounts of the cofactor but also avoids the accumulation of the reduced cofactor (NADPH) produced by morphine dehydrogenase and the consequent further reduction of hydromorphone to... 

The three branched chain amino acids are normally metabolized as shown in Figure 6.2. Each amino acid is converted to the corresponding Of-keto acid by a transaminase specific for that amino acid. A solitary case of valinaemia is known, caused by lack of valine transaminase [76] the patient is mentally retarded. The three a-keto acids are decarboxylated by two (or possibly three) enzyme systems, one specific for a-keto-isovaleric acid, the other acting on a-keto-isocaproic and Q -keto-j3-methylvaleric acids [77, 78]. The reaction is complex, proceeding in three distinct steps [78] and requiring coenzyme A, thiamine pyrophosphate, lipoic acid and NAD. The end products are the co-enzyme A thio-esters of the branched chain fatty acids. 

H)2-D3 is a weak agonist and must be modified by hydroxylation at position Cj for full biologic activity. This is accomplished in mitochondria of the renal proximal convoluted tubule by a three-component monooxygenase reaction that requires NADPFl, Mg, molecular oxygen, and at least three enzymes (1) a flavoprotein, renal ferredoxin reductase (2) an iron sulfur protein, renal ferredoxin and (3) cytochrome P450. This system produces l,25(OH)2-D3, which is the most potent namrally occurring metabolite of vitamin D. 

A system for describing kinetic mechanisms for enzyme-catalyzed reactions . Reactants (ie., substrates) are symbolized by the letters A, B, C, D, eto., whereas products are designated by P, Q, R, S, etc. Reaction schemes are also identified by the number of substrates and products utilized (i.e.. Uni (for one), Bi (two), Ter (three occasionally Tri), Quad (four), Quin (five), etc. Thus, a two-substrate, three-product enzyme-catalyzed reaction would be a Bi Ter system. In addition, reaction schemes are identified by the pattern of substrate addition to the enzyme s active site as well as the release of products. For a two-substrate, one-product scheme in which either substrate can bind to the free enzyme, the enzyme scheme is designated a random Bi Uni mechanism. If the substrates bind in a distinct order (note that, in such cases, A binds before B for ordered multiproduct release, P is released prior to Q, etc.), the scheme would be ordered Bi Uni. If the binding scheme is different than the release of product, then that information should also be provided for example, a two-substrate, two-product reaction in which the substrates bind to the enzyme in an ordered fashion whereas the products are released randomly would be designated ordered on, random off Bi Bi scheme. If one or more Theorell-Chance steps are present, that information is also given (e.g., ordered Bi Bi-(Theorell-Chance)), with the prefixes included if there is more than one Theorell-Chance step. 

The various ter-reactant enzyme systems can also be discriminated by the use of competitive inhibitors (see Table II). Fromm" has also presented a cogent argument for exercising special care in choosing the concentrations of the nonvaried substrates in experiments on reactions involving three substrates. 

The velocity of an enzyme-catalyzed reaction can be measured either by a continuous assay or by a stopped-time protocol. Whenever possible, the continuous measurement of a velocity (e.g., the increase or decrease in absorbance vx. time) should be utilized. In stopped-time assays, the investigator must demonstrate that the reaction is completely terminated at the specified point in time and that products are readily and quantitatively separated from substrates. In addition, one must show that the system is under initial rate conditions. Thus, at least three or four different time points should be chosen. Stopped-time assays also require an assay blank (for t = 0). In this blank, typically the quenching conditions are applied prior to the initiation step. Whenever practicable, replicate kinetic analyses should be done, even with continuous assay protocols. See Enzyme Assay Methods Basal Rate... 

Enzymes responsible for metabolism are located at various subcellular sites, for example the cytosol, mitochondria and smooth endoplasmic reticulum. However, it is enzymes derived from endoplasmic reticulum, called mixed function oxidases or monooxygenases , which have been most intensely studied in the past two or three decades. These enzyme systems, which utilize a family of haemoprotein cytochromes, or P-450 as terminal oxidases, require molecular oxygen and reduced nicotinamide adenine dinucleotide phosphate (NADPH) for activity. The overall stoichiometry of the reactions catalyzed by these enzymes is normally represented by equation (1). 

Reversible inhibition occurs rapidly in a system which is near its equilibrium point and its extent is dependent on the concentration of enzyme, inhibitor and substrate. It remains constant over the period when the initial reaction velocity studies are performed. In contrast, irreversible inhibition may increase with time. In simple single-substrate enzyme-catalysed reactions there are three main types of inhibition patterns involving reactions following the Michaelis-Menten equation competitive, uncompetitive and non-competitive inhibition. Competitive inhibition occurs when the inhibitor directly competes with the substrate in forming the enzyme complex. Uncompetitive inhibition involves the interaction of the inhibitor with only the enzyme-substrate complex, while non-competitive inhibition occurs when the inhibitor binds to either the enzyme or the enzyme-substrate complex without affecting the binding of the substrate. The kinetic modifications of the Michaelis-Menten equation associated with the various types of inhibition are shown below. The derivation of these equations is shown in Appendix S.S. 

In three of the reactions connecting the pools we find a big drop in free energy in the glycolytic direction (see table 12.1). Clearly if cells are to conduct these reactions in the reverse direction, ATP must be pumped into the system and different enzymes will be required. 

The parameter B2 is chosen for this project in order to gain some insight into possible consequences of varying the capability of the acetylcholinesterase to hydrolyze the neurotransmitter. Imbalances in this capability give rise to devastating diseases such as Alzheimer s and Parkinson s. The enzyme activity is included in the grouped parameter B2, which includes the maximum reaction velocity in reaction 2. The parameter B2 itself includes the enzyme activity together with three constants for the enzyme system, namely the concentration of acetylcholinesterase in compartment (II), the volume V2 of compartment (II), and the flow rate q. 

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