PLP-dependent enzyme

ACS isozyme utilizes pyridoxal-5 -phosphate (PLP) as a cofactor and belongs to fold type I PLP-dependent enzymes showing an absorption maximum between 422 and 431 nm, which is due to the internal aldimine. The reaction mechanism proposed for the conversion of SAM to ACC by ACS illustrated in Scheme 2 involves the following steps ... 

ACS activity may be reversibly regulated by various substances associated with the methionine-recycling pathway, SAM metabolism, and polyamine synthesis, and by natural and chemical analogues of SAM or inhibitors of PLP-dependent enzymes. 

The hydroxylamine or vinylglycine analogues are potent inhibitors of PLP-dependent enzymes in vivo and in vitro. The hydroxylamine analogues inactivate the enzyme forming stable oximes with PLP. 

Except the crystallographic asymmetric unit containing one molecule of ACS with AVG (HAY, 1M7Y), all other structures contain two molecules of ACS, which strongly suggests a functional dimeric form of these enzymes. In the dimeric form of both tomato and apple ACS, two independent active sites are formed at the interface of a dimer and share residues from each monomer. It has been well documented that the dimeric form is the basic catalytic unit of most PLP-dependent enzymes and that both monomers within a dimer physically share an active site." ... 

PLP-dependent enzymes catalyze the following types of reactions (1) loss of the ce-hydrogen as a proton, resulting in racemization (example alanine racemase), cyclization (example aminocyclopropane carboxylate synthase), or j8-elimation/replacement (example serine dehydratase) (2) loss of the a-carboxylate as carbon dioxide (example glutamate decarboxylase) (3) removal/replacement of a group by aldol cleavage (example threonine aldolase and (4) action via ketimine intermediates (example selenocysteine lyase). 

The product of the PNP enzyme, FDRP 9 has been purified and characterised. The evidence suggests that FDRP 9 is then isomerised to 5-fluoro-5-deoxyribulose-1-phosphate 10, acted upon by an isomerase (Scheme 7). Such ribulose phosphates are well-known products of aldolases and a reverse aldol reaction will clearly generate fluoroacetaldehyde 11. Fluoroacetaldehyde 11 is then converted after oxidation to FAc 1. We have also shown that there is a pyridoxal phosphate (PLP)-dependent enzyme which converts fluoroacetaldehyde 11 and L-threonine 12 to 4-FT 2 and acetaldehyde in a transaldol reaction as shown in Scheme 8. Thus, all of the biosynthetic steps from fluoride ion to FAc 1 and 4-FT 2 can be rationalised as illustrated in Scheme 7. 

Complexity of inhibition of PLP-dependent enzymes is highlighted by detailed investigations on the inhibition of y-aminobutyric acid aminotransferase (GABA-AT), the enzyme responsible for the degradation of y-aminobutyric acid (GABA), one of the major inhibitory neurotransmitters in the mammalian central nervous system. Inhibition of GAB A-AT results in an increased concentration of GABA in the brain and could have therapeutic applications in neurological disorders (epilepsy, Parkinson disease, and Alzheimer disease). 

Transamination (equation 12) requires two sets of reactions firstly, (34) is protonated at C-4 of PLP to yield a ketimine, the hydrolysis of which yields a carbohyl compound and PMP. The reverse reaction of PMP with a different carbonyl compound to that produced in the forward reaction accounts for the stoichiometry of equation (12). Transamination reactions mediated by PLP-dependent enzymes are widespread both in bacteria and in mammals. Over 50 different transaminases are known (B-73MI11000). 

Pyridoxal or PLP, in the complete absence of enzymes, not only undergoes slow transamination with amino acids but also catalyzes many other reactions of amino acids that are identical to those catalyzed by PLP-dependent enzymes. Thus, the coenzyme itself can be regarded as the active site of the enzymes and can be studied in nonenzymatic reactions. The latter can be thought of as models for corresponding enzymatic reactions. From such studies Snell and associates drew the following conclusions.148... 

PLP-dependent enzymes are inhibited by a great variety of enzyme-activated inhibitors that react by several distinctly different chemical mechanisms.11 Here are a few. The naturally occurring gabaculline mimics y-aminobutyrate (Gaba) and inhibits y-aminobutyrate aminotransferase as well as other PLP-dependent enzymes. The inhibitor follows the normal catalytic pathway as far as the ketimine. There, a proton is lost from the inhibitor permitting formation of a stable benzene ring and leaving the inhibitor stuck in the active site ... 

CHO group greatly enhances the catalytic activity Since certain metal ions, such as Cu2+ and Al3+, increase the rates in model systems and are known to chelate with Schiff bases of the type formed with PLP, it was concluded that either a metal ion or a proton formed a chelate ring and helped to hold the Schiff base in a planar conformation (Fig. 14-6). However, such a function for metal ions has not been found in PLP-dependent enzymes. 

In Fig. 14-5 the reactions of PLP-amino acid Schiff bases are compared with those of (i-oxo-acids. Beta-hydroxy-a-oxo acids and Schiff bases of PLP with (i-hydroxy-a-amino acids can react in similar ways. The reactions fall naturally into three groups (a,b,c) depending upon whether the bond cleaved is from the a-carbon of the substrate to the hydrogen atom, to the carboxyl group, or to the side chain. A fourth group of reactions of PLP-dependent enzymes (d) also involve removal of the a-hydrogen but are mechanistically more complex. Some of the many reactions catalyzed by these enzymes are listed in Table 14-3. 

The three-dimensional structures of these and other PLP-dependent enzymes were determined by 2000. 

Threonine is cleaved to acetaldehyde by the same enzyme. A related reaction is indicated in Fig. 24-27 (top). In a more important pathway of degradation of threonine the hydroxyl group of its side chain is dehydrogenated to form 2-amino-3-oxobutyrate which is cleaved by a PLP-dependent enzyme to glycine and acetyl-CoA (Eq. 14-31).214 215... 

Elimination reactions 526, 530, 677—690 beta, of cystine residues 85 conjugative 689 decarboxylative 689 facilitation by carbonyl group 681 of y substituent 746 of PLP-dependent enzymes 742 reversibility 690 Ellman s reagent 125,125s Elongation factor EF-Tu 558 Elongin complex 564... 

This is followed by ATP-dependent reduction to the aldehyde.2643 The final step of transamination is not accomplished in the usual way (with a PLP-dependent enzyme), but through formation of a Schiff base with glutamate and reduction to saccharopine.265 Oxidation now produces the Schiff base of lysine with 2-oxoglutarate. 

Lysine is not only a constituent of proteins. It can also be trimethylated and converted to carnitine (p. 944). In mammals some specific lysyl side chains of proteins undergo N-trimethylation and proteolytic degradation with release of free trimethyllysine (Eq. 24-30) 278/279 The free trimethyllysine then undergoes hydroxylation by a 2-oxoglutarate-Fe2+-ascorbate-dependent hydroxylase (Eq. 18-51) to form P-hydroxytrimethyllysine, which is cleaved by a PLP-dependent enzyme (Chapter 14). The resulting aldehyde is oxidized to the carboxylic acid and is converted by a second 2-oxoglutarate-Fe2+-ascorbate-dependent hydroxylase to carnitine (Eq. 24-30 see also Eq. 18-50). 

L-Serine is converted to pyruvate + NH3 by serine dehydratase (deaminase) in a PLP-dependent reaction. However, using the same coenzyme selenocysteine is converted by selenocysteine lyase into L-alanine + elemental selenium Se°. l-Cysteine may be converted by PLP-dependent enzymes into wither H2S or into S° for transfer into metal clusters. Compare the chemical mecha-... 

Alanine racemase, as another PLP-dependent enzyme, is a bacterial enzyme used to create D-alanine from L-alanine for incorporation into the bacterial cell wall. Its role is to act as an electron sink to stabilize carbanionic intermediates generated in enzymatic catalysis. 

AspAT is a PLP-dependent enzyme. This dimeric enzyme contains one PLP per subunit whose molecular weight is about 50,000. 

It is of interest to compare the tertiary structure of AspAT with that of other PLP-dependent enzymes. Some PLP enzymes whose primary structures are quite different from AspAT exhibit similar tertiary structures. Such enzymes are a>-amino acid pyruvate aminotransferase,341 phosphoserine aminotransferase351 and tyrosine-phenol lyase361 (Phillips, R., personal communication). Similarity in tertiary structure among these PLP enzymes may lead to the idea that many PLP-dependent enzymes share the same ancestor protein. There are PLP enzymes belonging to its own category, such as glycogen phosphorylase and tryptophan synthase.37 381 These enzymes do not share any similarities in either primary or tertiary structures with AspAT. 

Tryptophanase (L-tryptophan indole-lyase (deaminating) EC 4.1.99.1) belongs to the family of the pyridoxal 5 -phosphate (PLP)-dependent enzymes. It serves in vivo to degrade L-tryptophan, is induced by L-tryptophan, and found in various bacteria, particularly in enteric species. Tryptophanase catalyzes a,/3-elimination1 and /3-replacement reactions on interaction with L-tryptophan and various other /3-substituted amino acids2 ... 

Scheme VIII. Proposed two-base mechanism for amino acid racemization by PLP-dependent enzyme (according to [55]).

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