Multistep enzyme reactions

Several other successful applications of the low-temperature procedure to the thermal control and analysis of multistep enzyme reactions could be described. We prefer to cite appropriate papers (Douzou, 1974, 1977a,b Fink, 1976a) and to discuss two important problems raised by the present procedure, namely the validity of data obtained in such bizarre media and the necessity of obtaining suitable data on the conformational changes in proteins during their reaction pathways. 

Kinetic complexity definition, 43 Klinman s approach, 46 Kinetic isotope effects, 28 for 2,4,6-collidine, 31 a-secondary, 35 and coupled motion, 35, 40 in enzyme-catalyzed reactions, 35 as indicators of quantum tunneling, 70 in multistep enzymatic reactions, 44-45 normal temperature dependence, 37 Northrop notation, 45 Northrop s method of calculation, 55 rule of geometric mean, 36 secondary effects and transition state, 37 semiclassical treatment for hydrogen transfer,... 

We apply the concept of catalytic commitment, as proposed by Northrop, O Leary, and Cleland for multistep enzyme-catalyzed processes, to nonenzy-mic decarboxylation for comparison.52 The interpretation of CKIEs for decarboxylation reactions is dependent upon whether the process is viewed as a single-step or multi-step process. In a single-step mechanism, carbon-carbon bond-breaking is not affected by any other rate-limiting process. In this case, the CKIE for a particular compound will be constant under a standard set of conditions. Substantial changes in bond order must occur in the... 

Catechol melanin, a black pigment of plants, is a polymeric product formed by the oxidative polymerization of catechol. The formation route of catechol melanin (Eq. 5) is described as follows [33-37] At first, 3-(3, 4 -dihydroxyphe-nyl)-L-alanine (DOPA) is derived from tyrosine. It is oxidized to dopaquinone and forms dopachrome. 5,6-Dihydroxyindole is formed, accompanied by the elimination of C02. The oxidative coupling polymerization produces a melanin polymer whose primary structure contains 4,7-conjugated indole units, which exist as a three-dimensional irregular polymer similar to lignin. Multistep oxidation reactions and coupling reactions in the formation of catechol melanin are catalyzed by a copper enzyme such as tyrosinase. Tyrosinase is an oxidase con-... 

Studies of reaction mechanisms and enzymic reactions rely to a great extent on labeled adducts. The anticipated synthesis of the labeled precursors often are achieved only by multistep procedures performed chemically or enzymatically. Isotope-labeled dihydroneopterin 3 -triphosphate (51), with 3H at positions C-T and C-2, respectively, has been prepared from isotope-labeled glucose as starting material. 

This example of a classical cyclic resolution-racemization methodology indicates the limitations of a multistep enzyme-catalyzed reaction when the reaction medium, the reagents, or the reaction conditions are not mutually compatible. 

Several inventive procedures have been used for overcoming these difficulties, offering interesting multistep enzyme-catalyzed reactions for deracemization of amino acids. The requirement of an amino donor of D-configuration can be solved by its in situ generation from the L-form by using an amino acid racemase. 

The development of analytically useful enzyme electrodes is limited by the availability of purified and stable enzyme preparations. In an effort to extend the range of measurable species using ISE devices further, Rechnitz and co-workers (Rl) recently introduced bacterial- and tissue-based bio-selective electrode systems. These sensors are prepared in much the same manner as the enzyme probes except that whole intact cells are utilized as the immobilized reagents. There are several potential advantages to this novel approach, including (1) no need to extract and purify the enzymes involved, i. e., low cost (2) enzymes which are unstable when extracted from the cell may be used in situ to maximize and preserve their activity (3) if desired enzyme reactions require cofactors, these co ctors need not be added to the assay mixture because they are already present in the intact cell and (4) analytical reactions involving multistep enzyme sequences already present in the cells may be used to detect given analytes. 

This situation was presaged by Westheimer (1980), who stated ... that all enzymic reactions at phosphorus proceed with inversion and therefore they occur without pseudorotation at phosphorus. The argument for such a statement is based on two tenets first, the molecular motion caused by pseudorotation may require a conformational change of the enzyme to accommodate this movement secondly, a pseudorotation pathway will require a multistep mechanism. For example, the cyclization step of the reaction catalysed by ribonuclease may be postulated to involve pseudo-... 

Many reactions in biological chemistry (see also Biochemistry ) are multistep chemical reactions. Glycolysis, which is the process of breaking down sugar to generate energy, for example, involves ten sequential steps. Each step is carried out by a special type of catalyst, called an enzyme. There are countless multistep processes in biological systems. 

A detailed thermod5mamic analysis was performed with lactate dehydrogenase, in the lactate — p5mivate direction, by means of steady-state kinetics and presteady-state kinetic methods, by Laidler and Peterman (1979). A particularly detailed kinetic studies of the energetics of two multistep enzymes, triose-phosphate isomerase and prohne racemase, has been described by the research team of Albery and Knowles (Albery Knowles, 1976, 1986 Knowles, 1991). Apart from these examples, very few complete thermodynamic analyses have been performed with reactions involving more than one substrate or more than one intermediate in reaction. 

The presence in situ of biological structures supporting multicomponent or multistep biological reaction sequences useful for detection purposes and ensuring optimum enzyme activity (many enzymes lose activity when isolated in vitro)... 

Scheme 3.9 Designed biotransformation pathway starting from oleic acid yielding either n-nonanoic acid and oa-hydroxynonanoic acid or n-octanol and 1,10-decanedioic acid by multistep enzyme-catalyzed reactions.

Recently, multistep enzyme-catalyzed reactions have attracted the attention of chemists and biotechnologists, as they can be combined in a modular manner and often lead to high-value compounds. All naturally occurring metabolic pathways are basically cascade reactions. Based on natural principles, synthetic chemists search for universal multistep processes applicable to a vast number of chemical compounds. Multistep enzyme-catalyzed reactions involving nonphysiological substrates and selective enzymes are of particular interest because they may lead to tailor-made complex molecules with desired properties. Moreover, one of the most important advantages of multistep enzyme-catalyzed reaction sequences... 

The results of one of the first multistep enzyme systems to be immobilized were presented in 1970 by Mosbach and Mattiasson (3). They covalently bonded hexokinase (HK) and glucose-6-phosphate dehydrogenase (G-6-PDH) to individual polymer particles via the cyanogen bromide reaction. Using a solution containing glucose, ATP and NADP" ", they demonstrated that the coimmobilized enzymes formed product... 

These appHcations are mosdy examples of homogeneous catalysis. Coordination catalysts that are attached to polymers via phosphine, siloxy, or other side chains have also shown promise. The catalytic specificity is often modified by such immobilization. Metal enzymes are, from this point of view, anchored coordination catalysts immobilized by the protein chains. Even multistep syntheses are possible using alternating catalysts along polymer chains. Other polynuclear coordination species, such as the homopoly and heteropoly ions, also have appHcations in reaction catalysis. 

Biotransformations are carried out by either whole cells (microbial, plant, or animal) or by isolated enzymes. Both methods have advantages and disadvantages. In general, multistep transformations, such as hydroxylations of steroids, or the synthesis of amino acids, riboflavin, vitamins, and alkaloids that require the presence of several enzymes and cofactors are carried out by whole cells. Simple one- or two-step transformations, on the other hand, are usually carried out by isolated enzymes. Compared to fermentations, enzymatic reactions have a number of advantages including simple instmmentation reduced side reactions, easy control, and product isolation. 

The conversion occurs through a multistep sequence of reactions catalyzed by a complex of enzymes and cofactors called the pyruvate dehydrogenase complex. The process occurs in three stages, each catalyzed by one of the enzymes in the complex, as outlined in Figure 29.11 on page 1152. Acetyl CoA, the ultimate product, then acts as fuel for the final stage of catabolism, the citric acid cycle. All the steps have laboratory analogies. 

Figure 29.11 MECHANISM Mechanism of the conversion of pyruvate to acetyl CoA through a multistep sequence of reactions that requires three different enzymes and four different coenzymes. The individual steps are explained in the text.

Application of an aldolase to the synthesis of the tricyclic microbial elicitor (-)-syringolide (Figure 10.34) is another excellent example that enzyme-catalyzed aldolizations can be used to generate sufficient quantities of enantiopure material in multistep syntheses of complex natural and unnatural products [159]. Remarkably, the aldolase reaction established absolute and relative configuration of the only chiral centers that needed to be externally induced in the adduct (95) from achiral precursor (94) during the subsequent cyclization events, all others seemed to follow by kinetic preference. 

Nature, however, has performed more than simple stepwise transformations using a combination of enzymes in so-called multienzyme complexes, it performs multistep synthetic processes. A well-known example in this context is the biosynthesis of fatty acids. Thus, Nature can be quoted as the inventor of domino reactions. Usually, as has been described earlier in this book, domino processes are initiated by the application of an organic or inorganic reagent, or by thermal or photochemical treatment. The use of enzymes in a flask for initiating a domino reaction is a rather new development. One of the first examples for this type of reaction dates back to 1981 [3], although it should be noted that in 1976 a bio-triggered domino reaction was observed as an undesired side reaction by serendipity [4]. 

The immobilization of enzymes for sensing purposes frequently provides several important advantages an increase of its stability, operational reusability and greater efficiency in consecutive multistep reactions. Sometimes immobilization is accompanied by a certain degree of denaturalization however, the enzyme-matrix interactions may assist in stabilization preventing conformational transitions that favor such process. In some cases excessive bond formation affects the conformation of the active site and the steric hindrances caused by the polymer matrix may render an inactive sensor. 

Isotope effects have been used to determine whether the hydride transfer from the enzyme cofactor nicotinamide-adenine dinucleotide (NADH) (reaction (43)) takes place as a hydride ion transfer in a single kinetic step or in a multistep reaction via an uncoupled electron and hydrogen transfer. 

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