Pyridoxal phosphate replacements

This enzyme [EC 4.1.99.1], also known as L-tryptophan indole-lyase, catalyzes the hydrolysis of L-tryptophan to generate indole, pyruvate, and ammonia. The reaction requires pyridoxal phosphate and potassium ions. The enzyme can also catalyze the synthesis of tryptophan from indole and serine as well as catalyze 2,3-elimination and j8-replacement reactions of some indole-substituted tryptophan analogs of L-cysteine, L-serine, and other 3-substituted amino acids. 

Fig. 7.1 Reactions catalyzed at the active sites of the a subunit (a reaction) and of the 0 subunit (0 reaction) and the coupled, physiological reaction (a0 reaction). In the a0 reaction, indole produced by cleavage of indole-3-glycerol phosphate at the a site diffuses through an intramolecular tunnel to the 0 site 25-30 A distant where it undergoes a pyridoxal phosphate-dependent /3-replacement reaction with L-serine to form L-tryptophan. Abbreviations used IGP, indole-3-glyceroI phosphate G-3-P, o-glyceraldehyde 3-phosphate, IND, indole [IND], indole intermediate PLP, pyridoxal phosphate.

The chromophoric pyridoxal phosphate coenzyme provides a useful spectrophotometric probe of catalytic events and of conformational changes that occur at the pyridoxal phosphate site of the P subunit and of the aiPi complex. Tryptophan synthase belongs to a class of pyridoxal phosphate enzymes that catalyze /3-replacement and / -elimination reactions.3 The reactions proceed through a series of pyridoxal phosphate-substrate intermediates (Fig. 7.6) that have characteristic spectral properties. Steady-state and rapid kinetic studies of the P subunit and of the aiPi complex in solution have demonstrated the formation and disappearance of these intermediates.73-90 Fig. 7.7 illustrates the use of rapid-scanning stopped-flow UV-visible spectroscopy to investigate the effects of single amino acid substitutions in the a subunit on the rate of reactions of L-serine at the active site of the P subunit.89 Formation of enzyme-substrate intermediates has also been observed with the 012P2 complex in the crystalline state.91 ... 

Amino acid-1, the first substrate, combines with the enzyme, and its amino group is then transferred to the pyridoxal phosphate prosthetic group of the enzyme. The first product, a-keto acid-1, is released from the active site and is replaced by the second substrate, a-keto acid-2, to which the amino acid is then transferred from the enzyme to form amino acid-2. Amino acid-2 is then released from the active site of the enzyme, restoring the enzyme to its original form. 

Vitamin Be has a central role in the metabolism of amino acids in transaminase reactions (and hence the interconversion and catabolism of amino acids and the synthesis of nonessential amino acids), in decarboxylation to yield biologically active amines, and in a variety of elimination and replacement reactions. It is also the cofactor for glycogen phosphorylase and a variety of other enzymes. In addition, pyridoxal phosphate, the metabolically active vitamer, has a role in the modulation of steroid hormone action and the regulation of gene expression. 

The ring nitrogen of pyridoxal phosphate exerts a strong electron withdrawing effect on the aldimine, and this leads to weakening of all three bonds about the a-carbon of the substrate. In nonenzymic reactions, all the possible pyridoxal-catalyzed reactions are observed - a-decarboxylation, aminotrans-fer, racemization and side-chain elimination, and replacement reactions. By contrast, enzymes show specificity for the reaction pathway followed which bond is cleaved will depend on the orientation of the Schiff base relative to reactive groups of the catalytic site. As discussed in Section 9.3.1.5, reaction specificity is not complete, and a number of decarboxylases also undergo transamination. 

Aminomutases. The enzymes L-p-lysine mutase (which is also D-a-lysine mutase) and D-omithine mutase catalyze the transfer of an co-amino group to an adjacent carbon atom (Table 16-1). Two proteins are needed for the reaction pyridoxal phosphate is required and is apparently directly involved in the amino group migration. In the P-lysine mutase the 6-amino group of L-P-lysine replaces the pro-S hydrogen at C-5 but with inversion at C-5 to yield (3S, 5S)-... 

E. W. Miles, Pyridoxal phosphate enzymes catalyzing fl-elimination and 3-replacement reactions, in Pyridoxal Phosphate and Derivatives, Vol. I in the series Coenzymes and Cofactors, (eds. D. Dolphin, R. Poulson, and O. Avramovic), John Wiley and Sons, New York, 1986, pp. 253-310. 

Explain why the ability of PLP to catalyze an amino acid transformation is greatly reduced if the OH substituent of pyridoxal phosphate is replaced by an OCH3. 

The dehydration nd deamination may be effected through a Schiff s base formation with pyridoxal phosphate. Such a mechanism has been postulated by Metzler and Snell SS9). It will be noted in the discussion below that many of the dehydrases studied require pyridoxal-5-phosphate. Replacement of the hydroxyl-hydrogen atom of the substrate prevents deamination. 

FIGURE 16.3 Aminotransferase reactions, (a) Outline of the reaction, (b) Structures of the prosthetic group and parent compound, (c) Covalent linkage between enzyme and pyridoxal phosphate is replaced by covalent linkage between incoming amino acid and pyridoxal phosphate... 

Tryptophanase is an enzyme which catalyzes the stoichiometric interconversion of L-tryptophan and pyruvate, ammonia and indole it requires pyridoxal phosphate as a cofactor. The enzyme from Proteus rettgeri has been isolated in a crystalline state and its catalytic properties were investigated. The enzyme catalyzes a series of oc,p-elimination, P-replacement and the reversal of oc,P-elimination reactions. 

In a series of papers, Cook et al.60-63 presented results of the 31P NMR studies of pyridoxal 5 -phosphate dependent enzyme. O-acetylserine sulf-hydrylase is the enzyme which catalyses the final step of biosynthesis of l-cysteine, the replacement of p-acetoxy group of O-acetyl-L-serine by thiol [30] in bacteria and plants. 

In this transformation, l-G1u can be replaced by L-Asp, i-Om, or by other amino acids the reaction requires the presence of pyridoxal-5-phosphate (vitamin B ), or, thiamine (vitamin Bi) [39, 40], as coenzyme. 

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 ... 

Phosphonylation of a -3-(9-isopropylidene-a -pyridoxyl chloride via a Michaelis-Becker reaction, followed by deprotection with 1 M HCl and oxidation of the resulting primary alcohol with MnOj, produces diethyl (4-fonnyl-3-hydroxy-2-methyl-5-pyridyl)methylphosphonate (Scheme 5.38), an analogue of pyridoxal 5 -phosphate whose 5-position side chain has been replaced by a phospho-nomethyl group. The alcohol oxidation step can be accomplished with a wide range of reagents, such as activated MnOj in CHCI3 at room temperature (53%), PCC in CH2CI2 (83-86%), or the Swem reaction (>95%). ... 

Removal of the phosphate group from position 5 of the pyridoxal ring abolished the P2X" purinoceptor antagonist properties of pyridoxal-5-phosphate [54]. Replacement of the aldehyde moiety in pyridoxal-5-phosphate or PPADS with a CH2OH or CH2NH2 group, to... 

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