Pyridoxal phosphate reactivity

Ap4A, diadenosine tetraphosphate BBG, Brilliant blue green BzATP, 2 - 3 -0-(4-benzoyl-benzoyl)-ATP cAMP, cyclic AMP CCPA, chlorocyclopentyl adenosine CPA, cyclopentyl adenosine CTP, cytosine triphosphate DPCPX, 8-cyclopentyl-1,3-dipnopylxanthine IP3, inosine triphosphate lpsl, diinosine penta phosphate a,p-meATP, a,p-methylene ATP p.y-meATP, p.y-meihylene ATP 2-MeSADP, 2-methylthio ADP 2-MeSAMP, 2-methylthio AMP 2-MeSATP, 2-methylthio ATP NECA, 5 -W-ethylcarboxamido adenosine PPADS, pyridoxal-phosphate-6-azophenyl-2, 4 -disulfonic acid PLC, phospholipase C RB2, reactive blue 2 TNP-ATP, 2, 3 -0-(2,4,6-trinitrophenyl) ATP. 

Suicide Enzyme Inhibitors. Snicide substrates are irreversible enzyme inhibitors that bind covalently. The reactive anchoring group is catalytically activated by the enzyme itself through the enzyme-inhibitor complex. The enzyme thus produces its own inhibitor from an originally inactive compound, and is perceived to commit suicide. To design a substrate, the catalytic mechanism of the enzyme as well as the nature of the functional gronps at the enzyme active site must be known. Conversely, successful inhibition provides valuable information about the structure and mechanism of an enzyme. Componnds that form carbanions are especially usefnl in this regard. Pyridoxal phosphate-dependent enzymes form such carbanions readily becanse... 

Nucleophilic catalysis is a specific example of covalent catalysis the substrate is transiently modified by formation of a covalent bond with the catalyst to give a reactive intermediate. There are also many examples of electrophilic catalysis by covalent modification. It will be seen later that in the reactions of pyridoxal phosphate, Schiff base formation, and thiamine pyrophosphate, electrons are stabilized by delocalization. 

It is well over 40 years since Pfeiffer discovered that certain reactions of a-amino acid esters, in particular, ester exchange, racemization and oxygenation, are effected very readily when their Schiff bases with salicylaldehyde are complexed to a transition metal ion (most notably Cu11). The Schiff bases result from a condensation reaction between a reactive carbonyl group and the amino group of the amino acids. Snell and his co-workers43 were also one of the first to point out that similar reactions also occurred if pyridoxal was used instead of salicylaldehyde, and that there is a close analogy with pyridoxal phosphate-promoted enzymic reactions of a-amino acid metabolism. Since then much work has been due on these and other similar systems and their reactivities. 

Structures of vitamin B6 derivatives and the bonds cleaved or formed by the action of pyridoxal phosphate (a). The reactive part of the coenzyme is shown in red in (a). The bonds shown in red in (d) are the types of bonds in substrates that are subject to cleavage. 

Introduction of a reactive group adjacent to the reaction locus has proven to be a very effective approach to irreversible inhibitors of many pyridoxal phosphate (PLP) dependent... 

In a number of enzymes that catalyze reactions that might be assumed to be pyridoxal phosphate-dependent, pyruvate provides the reactive carbonyl group (Section 9.8.1). Other enzymes have reactive carbonyl groups provided by a variety of quinones. One of these quinones, pyrrolidone quino-linequinone, may be a dietary essential, although no mammalian enzymes... 

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. 

The result of this half-transaminase reaction is formation of pyridoxamine phosphate at the active site of the enzyme, and hence loss of activity. Pyridoxamine phosphate dissociates from the active site, so that if adequate pyridoxal phosphate is available the resultant apoenzyme can be reactivated. 

Studies on the reactivation of apoglycogen phosphorylase with a variety of analogs of pyridoxal phosphate have shown that the catalytic moiety is the 5 -phosphate group - only analogs with a reversibly protonatable dianion in this position have any activity In the nonactivated form of phosphorylase b, the phosphate is monoprotonated (-OPO3H ) when the enzyme has been activated, either allosterically or by phosphorylation (phosphorylase a), it is dianionic (-OPOa ). A glutamate residue in the active site acts as the proton acceptor or donor for this transition between the inactive and active forms of the cofactor. 

Katunuma and coworkers (1971) described a protease in the rat that hydrolyzes the apoenzymes of a number of pyridoxal phosphate-dependent enzymes it has no effect on other proteins or the holoenzymes. Presumably, it attacks the conserved amino acid sequence around the active lysine residue to which the internal Schiff base is formed. The activity ofthe enzyme is increased some 10- to 20-fold in vitamin Be deficiency, suggesting that its function is to degrade those enzymes that lose their coenzyme more readily, and so make more pyridoxal phosphate available for use by other enzymes. There is also evidence that some pyridoxal phosphate-dependent apoenzymes are modified to become incapable of activation by pyridoxal phosphate, although retaining immunological cross-reactivity with the normal form of the enzyme in vitamin Be deficiency (Nagata and Okada, 1985). 

As with the normal mechanism of the enzyme, the inactivation starts with Schiff base formation with the enzyme-bound pyridoxal phosphate, followed by removal of an a-proton by an active-site base to form the reactive electrophilic intermediate (82). This then partitions between hydrolysis of the Schiff base linkage, resulting in the keto product (83)and enzyme reactivation, and Michael-type addition of an enzyme active-site nucleophile, resulting in a stable covalently bonded enzyme adduct (84). 

The borohydride reduction of Schiff base formed between a protein and pyridoxal phosphate should be carried out at a mildly acidic pH. Although sodium borohydride is more unstable in acidic solution, this disadvantage is offset by the exceptional reactivity of the Schiff base salts which are formed in mildly acidic solution (pH 4.S-6.5) (Schellen-berg 1963). Reductions have been carried out after the protein and pyridoxal-5-phosphate have been incubated at a pH of 7.5 which is then changed to 4.5 or 6.5 with acetic acid (Rippa et al. 1967 Dempsey and Christensen 1962 Piszkiewicz et al. 1970) or after initial incubation at pH 6.0 (Schnackerz and Noltmann 1971). The relative merit of either... 

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