Multienzyme systems separate substrates

FIGURE 18.5 Schematic representation of types of multienzyme systems carrying out a metabolic pathway (a) Physically separate, soluble enzymes with diffusing intermediates, (b) A multienzyme complex. Substrate enters the complex, becomes covalently bound and then sequentially modified by enzymes Ei to E5 before product is released. No intermediates are free to diffuse away, (c) A membrane-bound multienzyme system. 

In one type of study, the HPLC assay method has been applied to the question of assaying the activity of two enzymes that can use the same substrate but form different products. In another, the HPLC method was applied to the case of related two different enzymes that can use the same substrate and form the same product. It was also applied to the case a single enzyme that can use either of two substrates to form two different products. In a separate application, the so-called reconstitution approach, a multienzyme pathway is reconstructed by the addition of enzymes one after the other to form a multienzyme complex and to thereby reconstitute a naturally occurring multienzyme complex. And finally, this approach allows us to use the HPLC method to study an intact, naturally occurring multienzyme system. 

It has also been demonstrated that expensive substrates such as UDP-Gal can be readily prepared in situ by enzymatic conversion of the relatively inexpensive sugar nucleotide uridine 5 -diphospho-a-D-glucopyranose (UDP-Glc) using a UDP-Gal 4-epimerase enzyme. This system, coupled with an appropriate UDP-Gal transferase, provides more economic access to enzymatically galactosylated compounds. In these multienzyme systems, to increase enzyme efficiency and also avoid multiple fermentations for separate enzyme preparations, fusion proteins have been constructed that contain both the Gal-epimerase and Gal-transferase enzymes. The use of these fused enzyme systems has increased in the recent years as their catalysis of sequential reactions can have a kinetic advantage over the mixture of two separated enzymes since the product of the first enzyme travels a shorter distance before being captured by the next enzyme in the sequence. 

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