Aminoacrylate, Schiff base

Nucleophilic groups from enzymes can add to double bonds, e.g., in an aminoacrylate Schiff base, or to multiple bonds present in die inhibitor. An example is y-vinyl y-aminobutyrate (4-amino-5-hexenoic acid), another inhibitor of brain y-aminobutyrate aminotransferase which is a useful anticonvulsant drug. 

Elimination of indole from the second quinonoid intermediate (Fig. 9.13, IV) results in formation of the aminoacrylate Schiff base intermediate (Fig. 9.13, V). The latter may either decompose to the final products completing the reaction of a, -elimination or may add a new reactive group (R-H, where R is a -SH, -SCH3, -OH, or -OCH3 group) at... 

In /3-replacement reactions, the /3-substituent of an amino acid substrate is replaced by a new /3-substituent. For the three enzymes (TRPS, OASS, and cystathionine /3-synthase (CBS)) whose mechanisms are discussed in this section, the catalytic reaction is composed of two distinct half-reactions. The /3-elimination is followed by a /3-addition where nucleophilic agents, indole, sulfide, and homocysteine, respectively, react with the ci-aminoacrylate Schiff base to form the final product, L-tryptophan, L-cysteine, and L-cystathionine, respectively. 

Indoleglycerol 3-phosphate (13) is converted into tryptophan (14) by the action of L-tryptophan synthase. The mechanism of this enzymatic reaction involves formation of a Schiff base with an enzyme-bound pyridoxal phosphate. The a-aminoacrylate Schiff base formed undergoes the addition of a p-substituent to produce tryptophan (Floss, 1986) (Fig. 7.5). 

The inactivation and labeling of L-aspartate-/ -decarboxylase probably involves nucleophilic attack by a group on the enzyme on the fl-carbon atom of the a-aminoacrylate-Schiff base formed in the interaction of 8-chloro-L-alanine and enzyme-bound pyridoxal 5 -phosphate. Labeling of the enzyme thus requires conditions under which the catalytic reaction can occur. An excess of yS-chloro-n-alanine is therefore required, since a substantial portion of the analog is converted to pyruvate before... 

Very detailed studies on the inhibition of alanine racemase by fluoroalanines have been conducted. This enzyme catalyzes the racemization of alanine to provide D-alanine, which is required for synthesis of the bacterial wall. This work has demonstrated that a more complex process than that represented in Figure 7.47 could intervene. For instance, in the case of monofluoroalanine, a second path (Figure 7.48, path b) occurs lysine-38 of the active site can also attack the Schiff base PLP-aminoacrylate that comes from the elimination of the fluorine atom. This enamine inactivation process (path b) has been confirmed by isolation and identification of the alkylation compound, after denaturation of the enzyme (Figure 7.48). ... 

Beta replacement is catalyzed by such enzymes of amino acid biosynthesis as tryptophan synthase (Chapter 25),184 O-acetylserine sulfhydrylase (cysteine synthase),185 186a and cystathionine (3-synthase (Chapter 24).187 188c In both elimination and (3 replacement an unsaturated Schiff base, usually of aminoacrylate or aminocrotonate, is a probable intermediate (Eq. 14-29). Conversion to the final products is usually assumed to be via hydrolysis to free aminoacrylate, tautomerization to an imino acid, and hydrolysis of the latter, e.g., to pyruvate and ammonium ion (Eq. 14-29). However, the observed stereospecific addition of a... 

A (3 replacement reaction catalyzed by the PLP-dependent tryptophan synthase converts indoleglycerol phosphate and serine to tryptophan. Tryptophan synthase from E. coli consists of two subunits associated as an a2P2 tetramer (Fig. 25-3). The a subunit catalyzes the cleavage (essentially a reverse aldol) of indoleglycerol phosphate to glyceraldehyde 3-phosphate and free indole (Fig. 25-2, step s).67 The P subunit contains PLP. It presumably generates, from serine, the Schiff base of aminoacrylate, as indicated in Fig. 25-2 (step f). The enzyme catalyzes the addition of the free indole to the Schiff base to form tryptophan. The indole must diffuse for a distance of 2.5 ran... 

There are several types of evidence that the L-serine derivative that activates the a reaction is the Schiff base formed between aminoacrylate and pyridoxal phosphate (ES III in Fig. 7.6). (1) Amino acids including l- or D-tryptophan and glycine that form tetrahedral,... 

P-Elimination and then replacement reaction of an a-amino acid with a nucleophile is very attractive from the viewpoint of synthetic organic chemistry because various P-substituted alanines may be prepared from a simple a-amino acid, such as serine, and nucleophiles. A reaction catalyzed by tryptophan synthase - the formation of tryptophan from serine and indole - is one of the most well-known P-elimination and replacement reactions (Scheme 2.7). Here, an aldimine Schiff base is derived from reaction of the enzyme-bound PLP with serine, which then dehydrates to give the Schiff base of PLP with 2-aminoacrylate. Indole then adds to the vinyl Schiff base, generating tryptophan after lysine aminolysis of the Schiff base product. 

The a subunit catalyzes the formation of indole from indole-3-glycerol phosphate, whereas each P subunit has a PLP-containing active site that catalyzes the condensation of indole and serine to form tryptophan. The overall three-dimensional structure of this enzyme is distinct from that of aspartate aminotransferase and the other PLP enzymes already discussed. Serine forms a Schiff base with this PLP, which is then dehydrated to give the Schiffbase of aminoacrylate. This reactive intermediate is attacked by indole to give tryptophan. 

In the absence of the nucleophilic substrate, the enzyme also exhibits a /3-elimination reaction, in which transimination of the a-aminoacrylate external Schiff base occurs giving free enzyme and free a-aminoacrylate, which decomposes in solution to pyruvate and ammonia. 

The mechanisms of these processes are closely related and appear to have in common the formation on the enzyme of an aminoacrylate-pyridoxal-P complex (Fig. 25, 1). Addition of a new nucleophile in a Michael fashion followed by hydrolysis of the Schiff base would represent an overall )8 replacement reaction, whereas hydrolysis of the aminoacrylate intermediate affords a-oxoacid and ammonia in a )S elimination reaction (see Fig. 25). In these reactions the crucial elimination process is straightforward when X is a good leaving group, but when X is an aromatic system (for example indolyl) a prior protonation of the aromatic nucleus is a prerequisite for elimination. 

First serine forms a protonated Schiff base (external aldimine) with pyridoxal-5 -phos-phate. Removal of the serine a-hydrogen leads to a quinonoid intermediate, which then can eliminate the (3-OH to generate the Schiff base of aminoacrylate. The reaction is com-... 

Cysteine synthesis in bacteria proceeds via a-aminoacrylic acid bound to the enzyme as a Schiff base with pyridoxal phosphate (Cook and Wedding, 1976). Partially purified cysteine synthase from spinach (Schmidt, 1977a) and Chlorella (Schmidt, 1977b) catalyzes an exchange of sulfide into cysteine, consistent with the above mechanism. The exchange of acetate into OAS that would be expected due to formation of the proposed enzyme intermediate was not tested. 

The results may be explained by the hypothesis that serine reacts with pyridoxal phosphate to form a Schiff base, that causes the labiliza-tion and subsequent dissociation of both the a-hydrogen and -hydroxyl group and removal as water (see Fig. 3, Chapter 14). The resulting product is an aminoacrylic acid derivative of pyridoxal phosphate enz3nne. Indole condenses at the double bond to form tryptophan upon dissociation from the enzyme. 

The biosynthesis of tryptophan occurs by condensation of L-serine with indole, this reaction is catalyzed by tryptophan synthetase. The enzyme is a pyridoxal-phosphate containing enzyme which catalyzes nucleophilic p-substitution reactions of amino acids. The p-hydroxyl group of serine is substituted by indole by the action of the enzyme. The reaction is thought to proceed via a ketimine intermediate (27) which undergoes elimination to give an aminoacrylate-pyridoxal phosphate Schiff base (28). Addition at the P-carbon of indole followed by reversal of the process constitutes the enzymatic synthesis of L-tryptophan. 

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