Cofactor incorporation

Biosynthesis of coen2yme A (CoA) ia mammalian cells incorporates pantothenic acid. Coen2yme A, an acyl group carrier, is a cofactor for various en2ymatic reactions and serves as either a hydrogen donor or an acceptor. Pantothenic acid is also a stmctural component of acyl carrier protein (AGP). AGP is an essential component of the fatty acid synthetase complex, and is therefore requited for fatty acid synthesis. Free pantothenic acid is isolated from hver, and is a pale yeUow, viscous, and hygroscopic oil. 

A lot of analytical techniques have been proposed in recent decades and most of them are based on enzymes, called dehydrogenases, which are not sensitive to oxygen and need cofactors such as NAD". The key problems which seriously hamper a wide commercialization of biosensors and enzymatic kits based on NAD-dependent enzymes are necessity to add exogenous cofactor (NAD" ) into the samples to be analyzed to incorporate into the biologically active membrane of sensors covalently bounded NAD" to supply the analytical technique by NAD -regeneration systems. 

Fluorouracil (5-fluorouracil, 5-FU, Fig. 5) represents an early example of rational drag design in that it originated from the observation that tumor cells, especially from gut, incorporate radiolabeled uracil more efficiently into DNA than normal cells. 5-FU is a fluorinated pyrimidine analog that must be activated metabolically. In the cells 5-FU is converted to 5-fluoro-2>deoxyuridine-monophosphate (FdUMP). This metabolite inhibits thymidilate synthase which catalyses the conversion of uridylate (dUMP) to thymidilate (dTMP) whereby methylenetetrahydrofo-late plays the role of the carbon-donating cofactor. The reduced folate cofactor occupies an allosteric site of... 

The high specificity required for the analysis of physiological fluids often necessitates the incorporation of permselective membranes between the sample and the sensor. A typical configuration is presented in Fig. 7, where the membrane system comprises three distinct layers. The outer membrane. A, which encounters the sample solution is indicated by the dashed lines. It most commonly serves to eliminate high molecular weight interferences, such as other enzymes and proteins. The substrate, S, and other small molecules are allowed to enter the enzyme layer, B, which typically consist of a gelatinous material or a porous solid support. The immobilized enzyme catalyzes the conversion of substrate, S, to product, P. The substrate, product or a cofactor may be the species detected electrochemically. In many cases the electrochemical sensor may be prone to interferences and a permselective membrane, C, is required. The response time and sensitivity of the enzyme electrode will depend on the rate of permeation through layers A, B and C the kinetics of enzymatic conversion as well as the charac-... 

An antimetabolite interferes with the normal cellular metabolites. For instance, it can act as an inhibitor of one or more enzymes whose substrates are metabolites. Others are incorporated into macromolecules instead of the metabolites. Development of antimetabolites exhibiting anti-cancer activity met with the greatest success for analogues of metabolites involved in the biosynthesis of nucleic acids and of cofactors containing nitrogenous bases. Compounds such as 5-fluorouracyl and methotrexate are remarkably effective against human cancers, even though they feature host toxicity. 

Since, in order to decarboxylate pyruvate, the cofactor (6-thioctic acid) must be in its oxidized form, Calvin suggested that, in the presence of light, the coenzyme shifts to the reduced (dithiol) form, thus markedly reducing the rate of incorporation of C14 into the Krebs cycle. Furthermore, Bradley and Calvin236(f) suggested that 6-thioctic acid is an acceptor of... 

An immobilized-enzyme continuous-flow reactor incorporating a continuous direct electrochemical regeneration of NAD + has been proposed. To retain the low molecular weight cofactor NADH/NAD+ within the reaction system, special hollow fibers (Dow ultrafilter UFb/HFU-1) with a molecular weight cut-off of 200 has been used [32],... 

As we will see in subsequent chapters, many metalloproteins have their metal centres located in organic cofactors (Lippard and Berg, 1994), such as the tetrapyrrole porphyrins and corrins, or in metal clusters, such as the Fe-S clusters in Fe-S proteins or the FeMo-cofactor of nitrogenase. Here we discuss briefly how metals are incorporated into porphyrins and corrins to form haem and other metallated tetrapyrroles, how Fe-S clusters are synthesized and how copper is inserted into superoxide dismutase. 

In the case of the DMSO reductase family, as pointed out above, the metal centre is bound to two molecules of the cofactor. DMSO reductase itself catalyses the reduction of dimethylsulfoxide to dimethylsulfide with incorporation of the oxygen atom of DMSO into water. The active site of the oxidized enzyme is an L2MoVI0(0-Ser) centre, which, upon reduction, loses the M=0 ligand to give a L2MoIV(0-Ser) centre. In the catalytic... 

At the same time, this redox lability makes Mo well suited as a cofactor in enzymes that catalyze redox reactions. An example is the prominence of Mo in nitrogen fixation. This prokaryotic metabolism, the dominant pathway for conversion of atmospheric Nj to biologically-useful NH, utilizes Mo (along with Fe) in the active site of the nitrogenase enzyme that catalyzes Nj reduction. Alternative nitrogenases that do not incorporate Mo have been identified, but are markedly less efficient (Miller and Eady 1988 Eady 1996). 

K. V. In vitro incorporation of nascent molybdenum cofactor into human sulfite oxidase, J Biol Chem 2001, 276, 1837-1844. 

For all these reasons, some chemical or genetical modifications have been applied into the binding sites of antibodies in order to improve their reactivity [22]. Antibodies can be modified by the incorporation of natural or synthetic catalysts into the antibody recognition site, as for instance transition metal complexes, cofactors, and bases or nucleophiles, to carry other catalytic functions, which open the way to... 

The vast majority of research focused on selenium in biology (primarily in the fields of molecular biology, cell biology, and biochemistry) over the past 20 years has centered on identification and characterization of specific selenoproteins, or proteins that contain selenium in the form of selenocysteine. In addition, studies to determine the unique machinery necessary for incorporation of a nonstandard amino acid (L-selenocysteine) during translation also have been central to our understanding of how cells can utilize this metalloid. This process has been studied in bacterial models (primarily Escherichia colt) and more recently in mammals in vitro cell culture and animal models). In this work, we will review the biosynthesis of selenoproteins in bacterial systems, and only briefly review what is currently known about parallel pathways in mammals, since a comprehensive review in this area has been recently published. Moreover, we summarize the global picture of the nonspecific and specific use of selenium from a broader perspective, one that includes lesser known pathways for selenium utilization into modified nucleosides in tRNA and a labile selenium cofactor. We also review recent research on newly identified mammalian selenoproteins and discuss their role in mammalian cell biology. 

Figure 1 Overview of specific use of seienium in bioiogical systems. Selenium can be incorporated into macromolecules in at least three separate pathways. From the reduced form of selenide, selenium is activated to selenophosphate by the action of the enzyme selenophosphate synthetase (SPS or SelD). This activated form is then used as a substrate for pathway-specific enzymes that lead to (1) insertion as selenocysteine into proteins during translation (selenoproteins), (2) incorporation into tRNA molecules as mnm Se U or Se U, and (3) insertion into a unique class of molybdoenzymes as a labile, but required, cofactor. The need for activation to selenophosphate has been demonstrated in all cases at the genetic and biochemical level, with the exception of the labile selenoenzymes, where activation of selenium has only been proposed based on proximity of genes within an operon encoding SPS and a molybdoenzyme. ...

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