Immobilized enzymes as tools for the demonstration of metabolon formation. A short overview

Beeckmans, Sonia; Driessche, Edilbert Van; Kanarek, Louis

doi:10.1002/jmr.300060408

Cited by 26 publications

(19 citation statements)

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“…What might be the significance of the second binding site still remains purely speculative. Since indications accumulate pointing to the existence of physical interactions between enzymes of the same metabolic pathway (31)(32)(33), one might speculate, e.g. on local conformational changes within the enzyme, resulting in stronger interactions with the neighboring enzymes.…”

Section: Discussionmentioning

confidence: 99%

Pig Heart Fumarase Contains Two Distinct Substrate-binding Sites Differing in Affinity

Beeckmans

Driessche

1998

Journal of Biological Chemistry

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A eukaryotic fumarase is for the first time unequivocally shown to contain two distinct substrate-binding sites. Pig heart fumarase is a tetrameric enzyme consisting of four identical subunits of 50 kDa each. Besides the true substrates L-malate and fumarate, the active sites (sites A) also bind their analogs D-malate and oxaloacetate, as well as the competitive inhibitor glycine. The additional binding sites (sites B) on the other hand also bind the substrates and their analogs D-malate and oxaloacetate, as well as L-aspartate which is not an inhibitor. Depending on the pH, the affinity of sites B for ligands (K d being in the millimolar range) is 1-2 orders of magnitude lower than the affinity of sites A (of which K d is in the micromolar range). However, saturating sites B results in an increase in the overall activity of the enzyme. The benzenetetracarboxyl compound pyromellitic acid displays very special properties. One molecule of this ligand is indeed able to bind into a site A and a site B at the same time. Four molecules of pyromellitic acid were found to bind per molecule fumarase, and the affinity of the enzyme for this ligand is very high (K d ‫؍‬ 0.6 to 2.2 M, depending on the pH). Experiments with this ligand turned out to be crucial in order to explain the results obtained. An essential tyrosine residue is found to be located in site A, whereas an essential methionine residue resides in or near site B. Upon limited proteolysis, a peptide of about 4 kDa is initially removed, probably at the C-terminal side; this degradation results in inactivation of the enzyme. Small local conformational changes in the enzyme are picked up by circular dichroism measurements in the near-UV region. This spectrum is built up of two tryptophanyl triplets, the first one of which is modified upon saturating the active sites (A), and the second one upon saturating the low affinity binding sites (B).Fumarase (fumarate hydratase, EC 4.2.1.2) catalyzes the reversible, stereospecific addition of water to fumarate to form L-malate (1). Being an enzyme of the citric acid cycle, it serves both catabolic and anabolic purposes and, as such, it is found within all living organisms. In eukaryotes, both mitochondrial fumarase (which is involved in the citric acid cycle) and the cytoplasmic enzyme are encoded by the same gene (2-4). These fumarase molecules are tetramers consisting of identical subunits of 50 kDa each, and their activity does not depend upon the presence of metal cations (1). In prokaryotes on the other hand, there are two distinct classes of fumarase molecules: class I fumarases are heat-labile and Fe 2ϩ -dependent, dimeric enzymes with subunits of 60 kDa each, having no obvious sequence homology to the eukaryotic enzymes, whereas class II fumarases are heat-stable, Fe 2ϩ -independent tetrameric enzymes with subunits of 50 kDa and showing extensive homology to the eukaryotic fumarases. Recently the three-dimensional structure of Escherichia coli class II fumarase has been unraveled (5-7). According to crystallograph...

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Section: Discussionmentioning

confidence: 99%

Pig Heart Fumarase Contains Two Distinct Substrate-binding Sites Differing in Affinity

Beeckmans

Driessche

1998

Journal of Biological Chemistry

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“…As was discussed by Beeckmans et al (1993), they should preferentially display similar physicochemical properties to the enzymes under investigation. We performed a migration experiment on free pig fumarase and the cytoplasmic isoenzymes of pig malate dehydrogenase and pig aspartate aminotransferase through immobilized isocitrate lyase and malate synthase.…”

Section: Specificity Of the Associationsmentioning

confidence: 99%

“…However, specific associations between enzymes of other metabolic pathways have been amply demonstrated and discussed in the past (see, Beeckmans et al, 1993;Clegg, 1984;Friedrich, 1986;Fulton, 1982;Keleti et al, 1989;Srere, 1987;Bernhard, 1986, 1987;Wombacher, 1983; for reviews on this topic). Such associations between consecutive enzymes of a pathway offer many advantages for the organism, such as: the possibility of passing around intermediates of the pathway without delivering them first to the bulk phase, thereby circumventing diffusion problems ; the possibility of channeling of intermediates within a path- way or towards different metabolic pathways, thereby allowing an immediate and flexible adaptation to the continuously changing needs of the organism; the creation of a special environment around the pathway, so that a high flux can be maintained with a moderate number of intermediate molecules ; protection of labile intermediates by reducing their life-times; extensively speeding up a number of processes by reducing the transient time (i.e.…”

Section: Final Conclusionmentioning

confidence: 99%

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A Specific Association Between the Glyoxylic‐Acid‐Cycle Enzymes Isocitrate Lyase and Malate Synthase

Beeckmans

Khan

Driessche

et al. 1994

European Journal of Biochemistry

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There is accumulating evidence that metabolic pathways are organized in vivo as multienzyme clusters or metabolons. To assess interactions between consecutive enzymes of a pathway in vitro, it is usually essential to modify the physical properties of water around the enzymes, e.g. by immobilizing the latter onto a solid support. Such immobilized enzyme preparations can be embedded in agarose gels and used for affinity electrophoresis [Beeckmans, S., Van Driessche, E. & Kanarek, L. Cell. Biochem. 43, In this study we use the aforementioned technique to investigate the association between two plant glyoxylic acid cycle enzymes, i.e. isocitrate lyase and malate synthase. A specific histochemical staining technique is described for both enzymes. Affinity electrophoresis using either isocitrate lyase or malate synthase as the immobilized enzyme clearly shows that associations are formed between both enzymes. Moreover, experiments with metabolically unrelated enzymes prove that the observed interaction is specific.In recent years it was realized that the aqueous-phase properties in the interior of a cell seem to be essentially different from those of an ordinary aqueous solution (Beeckmans et al

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“…Enzymes that function to form a complete process such as metabolic cycling are physically associated so as to ensure substrate channeling without diffusion limitations. This metabolic organization is intrinsic to cells and although whole cells may be visualized as a random mix of enzymes, recent studies suggest a specific intracellular organization of enzymes specifically to allow for channeling of enzyme intermediates (Beeckmans et al, 1993;). Many of the enzymes of the citric acid cycle, for example, are inhibited by their reactants, products, intermediates or even cofactors involved in the cycle; functioning as a continuous unit in which inhibitory intermediates are immediately removed, maintains enzyme functionality throughout the cycle.…”

Section: Introductionmentioning

confidence: 99%

Co-immobilized coupled enzyme systems in biotechnology

Betancor

Luckarift

2010

Biotechnology and Genetic Engineering Reviews

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Abbreviations: ATP-adenosine triphosphate; EDTA-ethylenediaminetetraacetic acid; CALB-Candida antarctica B lipase; GOX-glucose oxidase; HRP-horse radish peroxidase; ABTS-2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid); NADPH-Nicotinamide adenine dinucleotide phosphate reduced form; NADH-Nicotinamide adenine dinucleotide reduced form; CoA-Coenzyme A; FAD-flavin adenine dinucleotide; FADH2-flavin adenine dinucleotide reduced form; GTP-Guanosine-5'-triphosphate; GDPGuanosine-5'-diphosphate; DNA-Deoxyribonucleic acid Co-immobilized coupled enzyme systems in biotechnology AbstractThe development of coimmobilized multi-enzymatic systems is increasingly driven by economic and environmental constraints that provide an impetus to develop alternatives to conventional multistep synthetic methods. as in nature, enzyme-based systems work cooperatively to direct the formation of desired products within the defined compartmentalization of a cell. in an attempt to mimic biology, coimmobilization is intended to immobilize a number of sequential or cooperating biocatalysts on the same support to impart stability and enhance reaction kinetics by optimizing catalytic turnover.There are three primary reasons for the utilization of coimmobilized enzymes: to enhance the efficiency of one of the enzymes by the in-situ generation of its substrate, to simplify a process that is conventionally carried out in several steps and/or to eliminate undesired by-products of an enzymatic reaction. as such, coimmobilization provides benefits that span numerous biotechnological applications, from biosensing of molecules to cofactor recycling and to combination of multiple biocatalysts for the synthesis of valuable products.Biotechnology and Genetic Engineering Reviews -Vol. 27, 95-114 (2010) Co-immobilized coupled enzyme systems in biotechnology The development of coimmobilized multi-enzymatic systems is increasingly driven by economic and environmental constraints that provide an impetus to develop alternatives to conventional multistep synthetic methods. As in nature, enzyme-based systems work cooperatively to direct the formation of desired products within the defined compartmentalization of a cell. In an attempt to mimic biology, coimmobilization is intended to immobilize a number of sequential or cooperating biocatalysts on the same support to impart stability and enhance reaction kinetics by optimizing catalytic turnover.There are three primary reasons for the utilization of coimmobilized enzymes: to enhance the efficiency of one of the enzymes by the in-situ generation of its substrate, to simplify a process that is conventionally carried out in several steps and/or to eliminate undesired by-products of an enzymatic reaction. As such, coimmobilization provides benefits that span numerous biotechnological applications, from biosensing of molecules to cofactor recycling and to combination of multiple biocatalysts for the synthesis of valuable products.

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Immobilized enzymes as tools for the demonstration of metabolon formation. A short overview

Cited by 26 publications

References 84 publications

Pig Heart Fumarase Contains Two Distinct Substrate-binding Sites Differing in Affinity

Pig Heart Fumarase Contains Two Distinct Substrate-binding Sites Differing in Affinity

A Specific Association Between the Glyoxylic‐Acid‐Cycle Enzymes Isocitrate Lyase and Malate Synthase

Co-immobilized coupled enzyme systems in biotechnology

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