Lysine pyridoxal-5 -phosphate

The mechanism of the first part of transamination is shown in Figure 29.14. The process begins with reaction between the a-amino acid and pyridoxal phosphate, which is covalently bonded to the aminotransferase by an iminc linkage between the side-chain -NTI2 group of a lysine residue and the PLP aldehyde group. Deprotonation/reprotonation of the PLP-amino acid imine in steps 2 and 3 effects tautomerization of the imine C=N bond, and hydrolysis of the tautomerized imine in step 4 gives an -keto acid plus pyridoxamine... 

Figure 30-14. Catabolism of i-lysine. (a-KG, a-ketoglutarate Glu, glutamate PLP, pyridoxal phosphate.) Circled numerals indicate the probable sites of the metabolic defects in periodic hyperlysinemia with associated hyperammonemia and persistent hyperlysinemia without associated hyperammonemia.

Binding of pyridoxal phosphate to peptide PP-42 also appears to be selective for lysine 30. As was indicated by NMR spectroscopy and UV/vis experiments, only one of three potential lysine Schiff bases appeared to form. To determine the site or sites of attachment, the aldimine peptide intermediates were reduced, proteolytically cleaved, and the fragments analyzed by mass spectroscopy. This... 

Pyridoxal phosphate (4) is the most important coenzyme in amino acid metabolism. Its role in transamination reactions is discussed in detail on p. 178. Pyridoxal phosphate is also involved in other reactions involving amino acids, such as decarboxylations and dehydrations. The aldehyde form of pyridoxal phosphate shown here (left) is not generally found in free form. In the absence of substrates, the aldehyde group is covalently bound to the e-amino group of a lysine residue as aldimine ( Schiffs base ). Pyridoxamine phosphate (right) is an intermediate of transamination reactions. It reverts to the aldehyde form by reacting with 2-oxoacids (see p. 178). 

Biotin (5) is the coenzyme of the carboxylases. Like pyridoxal phosphate, it has an amide-type bond via the carboxyl group with a lysine residue of the carboxylase. This bond is catalyzed by a specific enzyme. Using ATP, biotin reacts with hydrogen carbonate (HCOa ) to form N-carboxybiotin. From this activated form, carbon dioxide (CO2) is then transferred to other molecules, into which a carboxyl group is introduced in this way. Examples of biotindependent reactions of this type include the formation of oxaloacetic acid from pyruvate (see p. 154) and the synthesis of malonyl-CoA from acetyl-CoA (see p. 162). 

In the absence of substrates, the aldehyde group of pyridoxal phosphate is covalently bound to a lysine residue of the transaminase (1). This type of compound is known as an aldimine or Schiffs base. During the reaction, amino acid 1 (A, la) displaces the lysine residue, and a new aldimine is formed (2). The double bond is then shifted by isomerization. 

This enzyme [EC 5.4.S.2] catalyzes the interconversion of lysine and (35 )-3,6-diaminohexanoate. The enzyme is stimulated by S-adenosylmethionine and pyridoxal phosphate. 

This enzyme [EC 2.6.1.36], also called lysine e-aminotransferase, catalyzes the pyridoxal-phosphate-depen-dent reaction of L-lysine with a-ketoglutarate (or, 2-ox-oglutarate) to produce 2-aminoadipate 6-semialdehyde... 

This pyridoxal-phosphate-dependent enzyme [EC 4.1.1.18] catalyzes the conversion of L-lysine to form cadaverine and carbon dioxide. 5-Hydroxy-L-lysine can also be decarboxylated by this enzyme. 

This pyridoxal-phosphate-dependent enzyme [EC 2.1.2.1], which has a recommended EC name of glycine hydroxymethyltransferase, catalyzes the reversible reaction of 5,10-methylenetetrahydrofolate with glycine and water to produce tetrahydrofolate and L-serine. The enzyme will also catalyze the reaction of glycine with acetaldehyde to form L-threonine as well as with 4-tri-methylammoniobutanal to form 3-hydioxy-N, N, N -trimethyl-L-lysine. 

The synthesis pathway of quinolizidine alkaloids is based on lysine conversion by enzymatic activity to cadaverine in exactly the same way as in the case of piperidine alkaloids. Certainly, in the relatively rich literature which attempts to explain quinolizidine alkaloid synthesis °, there are different experimental variants of this conversion. According to new experimental data, the conversion is achieved by coenzyme PLP (pyridoxal phosphate) activity, when the lysine is CO2 reduced. From cadeverine, via the activity of the diamine oxidase, Schiff base formation and four minor reactions (Aldol-type reaction, hydrolysis of imine to aldehyde/amine, oxidative reaction and again Schiff base formation), the pathway is divided into two directions. The subway synthesizes (—)-lupinine by two reductive steps, and the main synthesis stream goes via the Schiff base formation and coupling to the compound substrate, from which again the synthetic pathway divides to form (+)-lupanine synthesis and (—)-sparteine synthesis. From (—)-sparteine, the route by conversion to (+)-cytisine synthesis is open (Figure 51). Cytisine is an alkaloid with the pyridone nucleus. 

Amino groups are tunneled to glutamate from all amino acids except lysine and threonine. The enzymes are aminotransferases, and they are reversible. The two most important of these enzymes are alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Aminotransferases require pyridoxal phosphate as a coenzyme. The presence of elevated levels of aminotransferases in the plasma can be used to diagnose liver disease. 

Other enzymes in the aconitase family include isopropylmalate isomerase and homoaconitase enzymes functioning in the chain elongation pathways to leucine and lysine, both of which are pictured in Fig. 17-18.90 There are also iron-sulfur dehydratases, some of which may function by a mechanism similar to that of aconitase. Among these are the two fumarate hydratases, fumarases A and B, which are formed in place of fumarase C by cells of E. coli growing anaerobically.9192 Also related may be bacterial L-serine and L-threonine dehydratases. These function without the coenzyme pyridoxal phosphate (Chapter 14) but contain iron-sulfur centers.93-95 A lactyl-CoA... 

Figure 14-6 Drawing showing pyridoxal phosphate (shaded) and some surrounding protein structure in the active site of cytosolic aspartate aminotransferase. This is the low pH form of the enzyme with an N-protonated Schiff base linkage of lysine 258 to the PLP. The tryptophan 140 ring lies in front of the coenzyme. Several protons, labeled Ha, Hb, and Hd are represented in NMR spectra by distinct resonances whose chemical shifts are sensitive to changes in the active site.169...

Observation of an abnormally large shift in the position of fluorescent emission of pyridoxal phosphate (PLP) in glycogen phosphorylase answered an interesting chemical question.187188 A 330 nm (30,300 cm ) absorption band could be interpreted either as arising from an adduct of some enzyme functional group with the Schiff base of PLP and a lysine side chain (structure A) or as a nonionic tautomer of a Schiff base in a hydrophobic environment (structure B, Eq. 23-24). For structure A, the fluorescent emission would be expected at a position similar to that of pyridoxamine. On the other hand, Schiff bases of the... 

In addition to the above reagents, which modify specific tyrosine residues in the protein, desensitization has been reported with pyridoxal phosphate, which forms a Schiff base derivative with lysyl residues (43). This reagent was first reported by Marcus and Hubert (43) to react with FDPase from swine kidney and to abolish AMP sensitivity with very little loss of catalytic activity. With liver FDPase most of the sensitivity to AMP is lost when 7-8 residues are incorporated, with concomitant loss of about 25% of the enzymic activity (43). The effects become irreversible when the Schiff base derivative is reduced with NaBH4 and Are-pyridoxyllysine has been isolated from the reduced complex. In the presence of AMP the sensitive lysine residues are protected, but the amount of PLP incorporated is increased (43). 

Pyridoxal phosphate, a prosthetic group in glycogen phosphorylase, is covalently attached to a lysine side chain of the enzyme. The phosphate group of the pyridoxal phosphate probably acts as a general acid to transfer a proton to inorganic phosphate in the enzymatic mechanism. 

Besides a lysine 5,6-aminomutase, Clostridium sticklandii also has a D-ornithine 4,5-aminomutase (EC 5.4.3.5) [78, 79], D-Ornithine is generated from L-ornithine by ornithine racemase [80], The two genes encoding D-ornithine 4,5-aminomutase have been cloned, sequenced, and expressed in E. coli [81]. The enzyme is an a2/ 2-heterotetramer, consisting of 12 800 Da and 82 900 Da subunits. The protein requires Bn and pyridoxal phosphate as cofactors. Similar to the lysine 5,6-aminomutase, a conserved base-ofi)/histidine-on cobalamin binding motif is present in the 82 900 Da protein. 

Lysine mutase is the first of a group of AdoCbl-dependent enzymes that catalyses the 1,2-migration of an amino group (Fig. 26). It has been isolated from Clostridium sticklandii [39] and consists of a cobalamin-binding orange protein and a smaller yellow protein. Apart from AdoCbl, several other essential cofactors have been identified, such as pyridoxal phosphate, ATP, FAD, thiols, Mg2+ and K+ [38]. The function of the yellow protein and some of these cofactors is to renew continuously... 

A proportion of the vitamin Be in foods may be biologically unavailable after heating, as a result of the formation of (phospho)pyridoxyllysine by reduction of the alditnine (Schiff base) by which pyridoxal and the phosphate are bound to the e-amino groups of lysine residues in proteins. A proportion of this pyridoxyUysine may be useable, because it is a substrate for pyridoxamine phosphate oxidase to form pyridoxal and pyridoxal phosphate. However, it is also a vitamin Be antimetaboUte, and even at relatively low concentrations can accelerate the development of deficiency in experimental animals maintained on vitamin Be-deficient diets (Gregory, 1980a, 1980b). 

Pyridoxine phosphate oxidase is a flavoprotein, and activation of the erythrocyte apoenzyme by riboflavin 5 -phosphate in vitro can be used as an index of riboflavin nutritional status (Section 7.4.3). However, even in riboflavin deficiency, there is sufficient residual activity of pyridoxine phosphate oxidase to permit normal metabolism ofvitamin Be (Lakshmi and Bamji, 1974). Pyridoxine phosphate oxidase is inhibited by its product, pyridoxal phosphate, which binds a specific lysine residue in tbe enzyme. In tbe brain, tbe Ki of pyridoxal phosphate is of the order of 2 /xmol per L - the same as the brain concentration of free and loosely bound pyridoxal phosphate, suggesting that this inhibition may be a physiologically important mechanism in the control of tissue pyridoxal phosphate (Choi et al., 1987). 

Pyridoxine is rapidly converted to pyridoxal phosphate in the liver and other tissues. Pyridoxal phosphate does not cross cell membranes, and efflux of the vitamin from most tissues is as pyridoxal. Pyridoxal phosphate is exported from the liver bound to albumin by formation of a Schiff base to lysine (Zhang et al., 1999). Much of the free pyridoxal phosphate in the liver (i.e., that which is not protein bound) is hydrolyzed to pyridoxal, which is also exported, and circulates bound to both albumin and hemoglobin in erythrocytes. 

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