Pyridoxal phosphate-linked enzymes

The enzyme shows a high substrate specificity for AdoMet, and affinity to the substrate is also high with a Km ranging from 12 to 60 pM, and the pH optimum is between 8.5 and 9.5. Interestingly, S-adenosylethionine shows some activity as a substrate. The enzyme reaction is competitively inhibited by AVG and AOA, which are inhibitors of pyridoxal phosphate-linked enzymes. 

Gabaculine (3-amino-2,3-dihydrobenzoic acid) is a potent mechanism-based inhibitor of certain pyridoxal phosphate-linked enzymes. " " In plants, it is a potent inhibitor of chlorophyll biosynthesis the inhibition can be reversed by Gabaculine and another transaminase... 

We noted earlier in this chapter (Section II.C.3) the pyridoxal-phosphate-linked enzymatic route of synthesis of ACPC (9) from the a-aminobutyryl moiety of S-adenosylmethionine (99). ACPC (9) undergoes further metabolic processing by two distinct fragmentation routes, to a-ketobutyrate (138) and ammonia (equation 21) in bacteria and yeast or to ethylene (139) in fruit and other plant tissues (equation 22) where ethylene (139) is a potent hormone for fruit-ripening or wound-healing The bacterial enzyme, ACPC deaminase is much better characterized and is taken up first. 

Pyridoxal Phosphate-Linked Transaminases and Cleavage Enzymes... 

Pyridoxal phosphate is a required coenzyme for many enzyme-catalyzed reactions. Most of these reactions are associated with the metabolism of amino acids, including the decarboxylation reactions involved in the synthesis of the neurotransmitters dopamine and serotonin. In addition, pyridoxal phosphate is required for a key step in the synthesis of porphyrins, including the heme group that is an essential player in the transport of molecular oxygen by hemoglobin. Finally, pyridoxal phosphate-dependent reactions link amino acid metabolism to the citric acid cycle (chapter 16). 

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. 

In all cases the keto acids seem to be formed by typical a-ketogluta-rate-linked, pyridoxal phosphate-dependent transaminases (EC 2.6.1.6, etc.) (9, 154, 156, 157). There has been little study of isolated, presumably specific enzymes in connection with flavors, although the leucine and alanine aminotransferases of tomato have been precipitated with (NH4)oS04 (164, 165). Transaminase activity in Saccharomyces cere-visiae has a pH optimum of 7.2 (154), and a-ketoglutarate is the only amino group recipient (154, 166). Only aspartate and amino acids with hydrophobic side chains are acted on (154). 

Figure 21.6. Structure of Glycogen Phosphorylase. This enzyme forms a homodimer one subunit is shown in white and the other in yellow. Each catalytic site includes a pyridoxal-phosphate (PLP) group, linked to lysine 680 of the enzyme. The binding site for the phosphate (Pj) substrate is shown.

The collagen molecules formed by removal of the propeptides spontaneously assemble into fibrils. At this stage, the fibrils are still immature and lack tensile strength, which is acquired by cross-linking. The initial step in cross-link formation is the oxidative deamination of a-amino groups in certain lysyl and hydroxyly-syl residues catalyzed by lysyl oxidase. The enzyme is a copper-dependent (probably cupric) protein, and the reaction requires molecular oxygen and pyridoxal phosphate for full activity. Only native collagen fibrils function as substrates. 

Figure 21.6 Structure of glycogen phosphorylase. This enzyme forms a homodimer one subunit is shown in white and the other in yellow. Each catalytic site includes a pyridoxal phosphate PLP group, linked to lysine 680 of the enzyme. The binding site for the phosphate (Pj) substrate is shown. Not icp that the catalytic site lies between the C-termina domain and the glycogen-binding site, A narrow crevice, which binds four or five glucose units of glycogen, connects the two sites. The separation of the sites allows the catalytic site to phosphorolyze several glucose units before the enzyme must rebind the glycogen substrate. [Drawn from INOl.pdb.]...

This is an example of several enzymes in which an essential co-factor is covalently bound to the protein. For example, lipoic acid and biotin are covalently linked to the c-amino group of a specific lysine residue in certain enzymes. In some cases, pyridoxal phosphate is bound to the protein through the formation of a Schiff base involving the carbonyl group of the co-factor and an c-amino group of a lysine residue. In cytochrome c, the heme is attached through two thiol ether linkages to cysteine residues of the protein. 

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 ... 

Scheme 13.16. The initial steps in the formation of an Af-methyl-A -pyrrolium cation. Pyridoxal phosphate-catalyzed decarboxylation of ornithine (ornithine decarboxylase, EC 4.1.1.17) yields bntane-1,4-diamine (pntresdne). Ar nine (Arg,R) also nndergoes pyridoxal phosphate-catalyzed decarboxylation (arginme [Arg,R] decarboxylase,EC4.1.1.19) to agma-tine and then hydrolytic loss of nrea (agmatinase, EC 3.5.3.11) to prodnce the same diamine. Methylation on nitrogen by 5 -adenosylmethionine is catalyzed by pntrescine N-methyltransferase (EC 2.1.1.53). EC nnmbers and some graphic materials provided in this scheme have been taken from appropriate links in a URL starting with http7/ www.chem.qmul.ac.uk/iubmb/enzyme/.

A brilliantly simple and largely satisfying solution [16] to the observations on lysine and cadaverine incorporation has been proposed. It is consistent in particular with the observed incorporation of lysine with distinction between C-2 and C-6, loss of nitrogen from C-2 but retention of the C-2 proton and it allows for normal incorporation of cadaverine 6.26). Central to the proposal is enzyme-catalysed decarboxylation of lysine (lysine decarboxylase) and oxidation of cadaverine (diamine oxidase) both involving pyridoxal phosphate as coenzyme. The proposed sequence involves orthodox pyridoxal-linked intermediates of which 625) and 6.27) are common to both enzyme-mediated reactions (Scheme 6.8). It is an important... 

Having discussed the biosynthesis of pyridoxal phosphate and morphine in the preceding two sections, we ll end this chapter on natural-products chemistry by going up yet one more level in complexity and looking at polyketide biosynthesis. Unlike what happens in many metabolic pathways, where each separate step is catalyzed by a separate, relatively small enzyme, erythromycin and other polyketides are assembled by a single massive enzyme called a synthase. The synthase contains many enzyme domains linked together, with each domain catalyzing a specific biosynthetic step in sequence. 

Present data indicates that chain growth in both gramicidin S and the tyrocidines commences at the D-phenylalanine residue adjacent to proline and that the first step in the synthesis is the conversion of L-phenylalanine to D-phenyldanine. In the case of the tyrocidines, the synthesis then proceeds in order from the amino to the carboxyl terminus to form a linear decapeptide ending with a thiol ester linked leucine. The peptide then cyclises relatively slowly to the final product. Yamada and Kurahashi showed that the epimerisation of L-phenylalanine in the initial step did not require pyridoxal phosphate or FAD and they suggested that the reaction occurs via the thiol ester enzyme bound form (101), Figure 3.18. 

An clectromcric displacement of electrons from bunds a. b. or c (see diagram below) would result in the release ol a cation (H, R. urCOOH) and, sub.sequently. lead to the variety of reactions observed with pyridoxal. The extent tu which one of thc.se displacements predominates over others depends on the. structure of the amino acid and the environment (pH. solvent, catalysts, enzymes, and such). When this mechanism applies in vivo, the pyridoxal component is linked to the enzyme through the phosphate of the hydroxymethyl group. 

Alanine racemase is a bacterial enzyme that catalyzes racemization of l- and d-alanine, and requires pyridoxal 5 -phosphate (PLP) as a cofactor. The enzyme plays an important role in the bacterial growth by providing D-alanine, a central molecule in the peptidoglycan assembly and cross-linking, and has been purified from various sources15 161. The enzyme has been used for the production of stereospecifically deuterated NADH and various D-amino acids by combination of L-alanine dehydrogenase (E. C. 1.4.1.1), D-amino acid aminotransferase (E. C. 2.6.1.21), and formate dehydrogenase (E.C. 1.2.1.2)I17, 18. ... 

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