Multi-enzyme units

Nature gives us some illustrative examples of iterative methodologies in its biochemical mechanisms. The fatty acid-polyketide biosynthesis is one of them. The assembly of acyl units by sequential Claisen-type condensations to form a polyketide or fatty acid takes place at a multi-enzyme complex, at which the initial molecule is lengthened by one C2-unit per pass of a reaction cycle (Fig. 2). 

The biosynthesis of polyketides (including chain initiation, elongation, and termination processes) is catalyzed by large multi-enzyme complexes called polyketide synthases (PKSs). The polyketides are synthesized from starter units such as acetyl-CoA, propionyl-CoA, and other acyl-CoA units. Extender units such as malonyl-CoA and methylmalonyl-CoA are repetitively added via a decarboxylative process to a growing carbon chain. Ultimately, the polyketide chain is released from the PKS by cleavage of the thioester, usually accompanied by chain cyclization [49]. 

PDH is a multi-enzyme complex consisting of three separate enzyme units pyruvate decarboxylase, transacetylase and dihydrolipoyl dehydrogenase. Serine residues within the decarboxylase subunit are the target for a kinase which causes inhibition of the PDH the inhibition can be rescued by a phosphatase. The PDH kinase (PDH-K) is itself activated, and the phosphatase reciprocally inhibited, by NADH and acetyl-CoA. Figure 3.12(a and b) show the role and control of PDH. 

A Fatty acids are constmcted by stepwise addition of two-carbon units by a large multi-enzyme complex located in the cytoplasm of all cells. 

C. Fatty acid synthase is a large multi-enzyme complex that catalyzes the addition of two-carbon units in a seven-step cycle (Figure 8-2). 

At the start of optimization of the reaction system, suitable values for pH and temperature have to be chosen as a function of the properties of the reactants and enzymes. Fortunately, most enzyme reactions operate in a narrow band with respect to pH value (7-10) and temperature (30-50 °C). The initial substrate concentration and, in the case of two-substrate reactions, the stoichiometric ratio of the two reactants, have to be selected. The selected enzyme concentration influences both the achievable space-time-yield as well as the selectivity in the case of undesired parallel or consecutive side reactions. In the case of multi-enzyme systems, the optimal activity ratio has to be found. The activity and stability of all the enzymes involved have to be known as a function of the reaction conditions, before the kinetic measurements are made. Enzyme stability is an important aspect of biocatalytic processes and should be expressed preferably as an enzyme unit consumption number, with the dimension unit of activity per mass of product (such as mole, lb, or kg). In multi-enzyme systems the stability of all the enzymes has to be optimized so that an optimal reaction rate and space-time-yield result. 

Table III. Specific enzyme activities of multi-enzyme complexes in technical fungal pectinase preparations expressed as International Units per g of Enzyme Preparation...

TA is also an enzyme of the oxidative pentose phosphate pathway[218). It catalyzes the transfer of the C1-C3 aldol unit from D-sedoheptulose 7-phosphate to D-Gly 3-P, and produces D-Fru 6-P and D-erythrose 4-phosphate (Fig. 14.2-3). TA forms a Schiffbase intermediate and does not require any co-factors. This enzyme is commercially available, and was used in a multi-enzyme synthesis of D-Fru from starch (Fig. 14.2-4) 1233] Here, it accomplished transfer of an aldol moiety from Fru 6-P to d-glyceraldehyde, and formed D-Gly 3-P and D-Fru. 

Oxoglutarate undergoes oxidative decarboxylation to succinyl-CoA, via multi-enzyme reaction similar to the reaction pattern of pyruvate. The multi-enzyme complex (mw about 2 x 10 ) is an octamer of an elementary unit containing each of the three contributing enzyme proteins oxoglutarate decarboxylase, dihydro-lipoyl transacetylase, and dihydrolipoyl dehydrogenase. The overall reaction involves thiamine pyrophosphate, lipoic acid, CoASH and NAD succinyl-CoA is the end product ... 

The practicability of such cyclic multi-enzyme preparations has been confirmed, e.g. for the synthesis of A -acetyllactosamine or the tetrasaccharide, sialyl Lewis, containing four different monosaccharide units. 

Since the components of each set were eluted by the same hapten group, galactose or lactose, and each protein combines with the same structural unit of the antigen, the individual proteins of each set have been designated as isoantibodies. This definition is a more restrictive definition of an isoantibody than is employed by immunologists (32) but is in line with the terminology employed by enzymologists for multi-molecular forms of enzymes (33). 

The second main class of blood constituents used as genetic markers are the polymorphic enzymes. The enzymes of interest to the forensic serologist are primarily located within the red blood cell and are commonly referred to as isoenzymes. These can briefly be described as those enzymatically active proteins which catalyze the same biochemical reactions and occur in the same species but differ in certain of their physicochemical properties. (This description does not exclude the tissue isoenzymes that occur within the same organism however, our consideration deals only with those of the red blood cell in particular.) The occurrence of multi-molecular forms of the same enzyme (isoenzymes) has been known for several decades however, it was not until the Metropolitan Police Laboratory of Scotland Yard adapted electrophoretic techniques to dried blood analysis that these systems were catapulted to the prominence they presently receive (.2). For many of the forensic serologists in the United States, the use of electrophoresis and isoenzyme determination is a recently-inherited capability shared by only a few laboratories. 

This is becoming known by the above name but the name used in the seminal papers of Curme et al. and Spayd et al. of Eastman Kodak Company is multi layer film elements for clinical analysis 44-45). It is a technique which unites two aspects of clinical analysis. One is the procedure for carrying out quantitative tests on fibre strips. These strips may be dipped into a sample of urine and instantly reveal the concentration of up to 8 basic components it is an elegant variation of indicator paper. The other aspect is the increasing use being made of biological methods, particularly enzyme methods for clinical analysis. 

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