Multistep enzyme systems

Mosbach K and Mattiasson B 1976 Multistep enzyme systems Methods in Enzymology vol 44, ed K Mosbach (New York Academic) pp 453-79 Olsson B, Lundback H and Johansson G 1985 Galactose determination in an automated flow-injection system containing enzyme reactors and an on-line dialyzer A a/. Chim. Acta 167 123-36... 

Co-enzyme A Multistep enzyme system (Brevibaclerium ammoniagenes) 115 (95)... 

It is the purpose of this paper to review the existing state of knowledge on gel entrapped multistep enzyme systems with particular reference to their kinetic behavior, and to add some recent results from this laboratory. 

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... 

The quantitative data and observations discussed above appear to be consistent with the qualitative concepts put forward by Mosbach and others to explain the behavior of multistep enzyme systems. Mathematical modelling of gel entrapped, multistep, immobilized enzyme systems, using the collocation technique, is quite straightforward and provides the opportunity not only to compare experiment with theory but also to explore the effect of parameters of the system which are experimentally inaccessible. 

Figure 2. A multistep enzyme interconversion cascade illustrating the sequential modification and conversion of target enzymes from one state of activity to another. Although written here as a cascade of increasing catalytic activity, enzyme-catalyzed covalent modification can either activate or inhibit target enzymes, depending on the particular system under study.

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. 

The immobilization of whole cells provides a means for the entrapment of multistep and cooperative enzyme system present in the intact cell, repetitive use and improved stabihty. This technique is also advantageous in the separation of bioproducts from cell mass in a continuous bioconversion process [114,115]. The other advantages of immobilized growing cells include (1) protection of cells against unfavourable environmental factors (2) changes in the permeability of the cells (3) reduced inhibition by substrate and product (4) reusability and (5) faster removal of end product. 

Figure 10.1 ThecyclicAMPsecondmessengersystem.Themostcommonsecond messenger system activated by the protein/peptide hormones and the catecholamines involves the formation of cAMP. This multistep process is initiated by binding of the hormone (the first messenger) to its receptor on the cell surface. The subsequent increase in the formation of cAMP (the second messenger) leads to the alteration of enzyme activity within the cell. A change in the activity of these enzymes alters cellular metabolism.

The anticoagulant fondaparinux, a synthetic analogue of the terminal fragment of heparin, is synthesized using multiple protection/deprotection steps that result in a route of up to 50 steps. There is, as yet, no enzymatic system that approaches the capability to make such a molecule." As this modified pentasaccharide is a natural product, it should, in theory, be accessible through a series of biotransformations, but we currently lack the biocatalytic tools to achieve more than a few steps and would stiU need to use some protection steps to avoid multiple products. Enzymatic synthesis in vivo depends largely on the levels and selectivities of glycosylating enzymes to achieve multistep reactions, a situation that has been mimicked in vitro for simpler systems." ... 

Experiments with chloroplasts showed an apparent inhibition of fatty acid synthesis by PAN (at 72 ppm for 10 min). The result is difficult to interpret the inhibition could be attributed to inactivation of one of the enzymes of the multistep system or to oxidation of the reductant (reduced NADP, or NADPH) required in the chain elongation process. 

A mathematical simplification of rate behavior of a multistep chemical process assuming that over a period of time a system displays little or no change in the con-centration(s) of intermediate species (i.e., d[intermedi-ate]/df 0). In enzyme kinetics, the steady-state assumption allows one to write and solve the differential equations defining fhe rafes of inferconversion of various enzyme species. This is especially useful in initial rate studies. 

MECHANISM FIGURE 22-18 Tryptophan synthase reaction. This enzyme catalyzes a multistep reaction with several types of chemical rearrangements. An aldol cleavage produces indole and glyceraldehyde 3-phosphate this reaction does not require PLP. Dehydration of serine forms a PLP-aminoacrylate intermediate. In steps and this condenses with indole, and the product is hydrolyzed to release tryptophan. These PLP-facilitated transformations occur at the /3 carbon (C-3) of the amino acid, as opposed to the a-carbon reactions described in Figure 18-6. The /3 carbon of serine is attached to the indole ring system. Tryptophan Synthase Mechanism... 

The simplest enzymatic system is the conversion of a single substrate to a single product. Even this straightforward case involves a minimum of three steps binding of the substrate by the enzyme, conversion of the substrate to the product, and release of the product by the enzyme (Scheme 4.6). Each step has its own forward and reverse rate constant. Based on the induced fit hypothesis, the binding step alone can involve multiple distinct steps. The substrate-to-product reaction is also typically a multistep reaction. Kinetically, the most important step is the rate-determining step, which limits the rate of conversion. 

The immobilization concept was later extended and applied to living cells41 . Immobilization of whole cells rather than purified enzymes reduced the expense of separation, isolation and purification of the enzyme. Furthermore, in multistep reactions, in which several enzymes are involved, the application of immobilized cells is advantageous. Since the enzymes are in their native state their stability is enhanced. Such systems may widely be applied, which is not possible with isolated pure enzymes, and are less expensive than processes based on free intact cells 42). 

This work on organic reaction mechanisms and our development of cyclophane chemistry were of great use to us in our later work. We did not shy away from tackling either multistep syntheses (up to 30 reactions) or highly asymmetric, designed systems needed in our studies of enzyme-mimicking systems. We needed both equilibria and kinetic techniques and an understanding of the importance of solvent effects in our more recent studies. 

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