Pyridoxal 5 -phosphate nucleotides

Hydrolases represent a significant class of therapeutic enzymes [Enzyme Commission (EC) 3.1—3.11] (14) (Table 1). Another group of enzymes with pharmacological uses has budt-ia cofactors, eg, in the form of pyridoxal phosphate, flavin nucleotides, or zinc (15). The synthases, and other multisubstrate enzymes that require high energy phosphates, are seldom available for use as dmgs because the required co-substrates are either absent from the extracellular space or are present ia prohibitively low coaceatratioas. 

The amino acid and nucleotide biosynthetic pathways make repeated use of the biological cofactors pyridoxal phosphate, tetrahydrofolate, and A-adenosylmethionine. Pyridoxal phosphate is required for transamination reactions involving glutamate and for other amino acid transformations. One-carbon transfers require S-adenosyhnethionine and tetrahydrofolate. Glutamine amidotransferases catalyze reactions that incorporate nitrogen derived from glutamine. 

Hydrolases represent a significant classes of therapeutic enzymes (Table 11. Another group of enzymes with pharmacological uses has built-in cofactors, c.g.. in the form of pyridoxal phosphate. Ilavin nucleotides, or zinc. 

Tissue also contains some endogenous species that exhibit fluorescence, such as aromatic amino acids present in proteins (phenylalanine, tyrosine, and tryptophan), pyridine nucleotide enzyme cofactors (e.g., oxidized nicotinamide adenine dinucleotide, NADH pyridoxal phosphate flavin adenine dinucleotide, FAD), and cross-links between the collagen and the elastin in extracellular matrix.100 These typically possess excitation maxima in the ultraviolet, short natural lifetimes, and low quantum yields (see Table 10.1 for examples), but their characteristics strongly depend on whether they are bound to proteins. Excitation of these molecules would elicit background emission that would contaminate the emission due to implanted sensors, resulting in baseline offsets or even major spectral shifts in extreme cases therefore, it is necessary to carefully select fluorophores for implants. It is also noteworthy that the lifetimes are fairly short, such that use of longer lifetime emitters in sensors would allow lifetime-resolved measurements to extract sensor emission from overriding tissue fluorescence. 

Fig. 8.1. Two-dimensional schematic representation of the structure of the adenine nucleotide carrier. The line represents the amino acid chain of the protein and all numbers on or within the line represent the number of the amino acids in the linear sequence. The black dots are cysteine residues, about which there is significant sequence homology [186]. The helical regions are segments of hydrophobic amino acids thought to span the membrane [186]. The CAT arrow represents the site of photoaffinity labelling of an azido derivative of atractyloside [189]. The open circles are lysine residues which react with pyridoxal phosphate in intact mitochondria or submitochondrial particles [190,191].

Fig. 38.5. Summary of the sources of NH4 for the urea cycle. All of the reactions are irreversible except glutamate dehydrogenase (GDH). Only the dehydratase reactions, which produce NH4 from serine and threonine, require pyridoxal phosphate as a cofactor. The reactions that are not shown occurring in the muscle or the gut can all occur in the liver, where the NH4 generated can be converted to urea. The purine nucleotide cycle of the brain and muscle is further described in Chapter 41.

Unspecific inhibition of ribonucleotide reduction is produced by compounds like pyridoxal phosphate, or the sulfonated anthraquinone-triazine dye, Cibacron blue. They interact, like in many enzymes, with nucleotide binding domains where pyridoxal phosphate becomes covalently linked to lysine, or in that the dye occupies the whole nucleotide fold. The latter interaction permits its use in affinity chromatography of ribonucleotide reductases Likewise, EDTA is not a specific, nor a potent inhibitor, it may, for example, act by complexation of the structure-stabilizing Mg " ions in native holoenzymes. However the iron-promoted radical regeneration process appears far more susceptible to interference from EDTA ... 

Although the 20 amino acids and complex folding patterns open up a range of chemical possibilities to proteins, there are also many limitations. In order to widen the range of chemistry available to enzyme catalysts, we make use of auxiliary compounds particularly well suited to certain types of reaction (Fig. A6.1). Thus NAD+, NADP+ and FAD are nucleotide cofactors based on the stmctures of nicotinamide and riboflavin, and are used for oxidation-reduction reactions. Biotin is used for carboxylation, pyridoxal phosphate is used for transamination and decarboxylation and so on. As mentioned in Box 3 (Topic 10), most of these are derived from vitamins in the diet. 

Thiol enzyme. SH-enzyme an enzyme whose activity depends on the presence of a certain number of free tUol groups. T.e. are found among the hydrolases, oxidoreductases and transferases. Known T.e. are bromelain, papain, urease, various flavoenzymes, pyridine nucleotide enzymes, pyridoxal phosphate enzymes and thiolproteinases. T.e. are t ically inhibited by Sulfhydryl reagents (see). 

High resolution studies " on phosphorylase b have permitted the binding sites of the substrate (o-glucose-l-phosphate), an inhibitor (D-glucose-6-phosphate), and the allosteric effectors adenosine mono- and di-phosphates (AMP and ATP) to be established. For this protein, which is an important example of allosteric control, it was determined that the D-glucose-1-phosphate was bound to a site about 8 A from the pyridoxal phosphate (which is an essential cofactor) and 33 A from the region which binds the adenosine nucleotides. 

Although the nicotinamide nucleotide coenzymes function in a large number of oxidation and reduction reactions, this cannot be exploited as a means of assessing the state of the body s niacin reserves, because the coenzymes are not firmly attached to their apoenzymes, as are thiamin pyrophosphate, riboflavin and pyridoxal phosphate. 

Nicotinic acid and riboflavin, the precursors of the pyridine and flavin nucleotides and ubiquinone or coenzyme Q, are essential components of the electron-transport chain. Pyridoxine, the precursor of pyridoxal phosphate, is essential for the activity of transaminases and amino acid decarboxylases and, therefore, for gluconeogenesis and for the production of biogenic amines. 

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