A-aminoacrylate intermediate

Reaction of the Tryptophan Synthase a-Aminoacrylate Intermediate with Indole... 

REACTION OF THE TRYPTOPHAN SYNTHASE a-AMINOACRYLATE INTERMEDIATE WITH INDOLE... 

MECHANISM FIGURE 22-18 Tryptophan synthase reaction. This enzyme catalyzes a multistep reaction with several types of chemical rearrangements. An aldol cleavage produces indole and glyceraldehyde 3-phosphate this reaction does not require PLP. Dehydration of serine forms a PLP-aminoacrylate intermediate. In steps and this condenses with indole, and the product is hydrolyzed to release tryptophan. These PLP-facilitated transformations occur at the /3 carbon (C-3) of the amino acid, as opposed to the a-carbon reactions described in Figure 18-6. The /3 carbon of serine is attached to the indole ring system. Tryptophan Synthase Mechanism... 

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... 

Investigations of the mechanism of the a/3 reaction by steady-state and transient kinetic methods have determined the rate constants for intermediate steps in the reaction.I04 The transient kinetic results show that diffusion of indole and condensation of indole with the aminoacrylate intermediate (ES III in Fig. 7.6) are rapid steps that occur without a lag reprotonation of the nascent tryptophan carbanion (ES IV in Fig. 7.6) is the rate-limiting step. The data rule out diffusion of free indole through the bulk solvent and support the channeling mechanism. 

Cook, P.F., Tai, C.H., Hwang, C.C., Woehl, E.U., Dunn, M.F., and Schnackerz, K.D. (1996) Substitution of pyridoxal 5 -phosphate in the O-acetylserine sulfhydrylase from Salmonella typhimurium by cofactor analogs provides a test of the mechanism proposed for formation of the alpha-aminoacrylate intermediate. J. Biol. Chem. 271, 25842-25849. 

C-Glycopyranosides may be obtained from glycopyranosyl halides via intermolecular addition of glycopyranosyl radicals [129]. In a more useful example, the a-aminoacrylate 192 was used as the radical acceptor for preparation of C-glycosyl amino acids 193 and 194 [130] (Scheme 66). In a concise synthesis of showdomycin (197), Barton utilized the trigger reaction of the 7V-hydroxy-2-thiopyridone derivative and the exceptional radicophilicity of tellurides in concocting the conditions for the conversion from the anisyl telluride 195 to the intermediate 196 after oxidative elimination [131] (Scheme 67). In Keck s synthesis of (-t-)-pseudomonic acid C (201), the intermediate 200 was prepared via stereocontrolled intermolecular addition of the radical generated from the iodide 198 to the allylic sulfone 199 [132] (Scheme 68). 

Catalysis by some, if not all, of these enzymes appears to involve intramolecular proton transfers mediated by a single active site base. In the case of the trypto-phanase reaction, an intramolecular 1,3-proton transfer takes place between C-2 of tryptophan and C-3 of the indol leaving group, on the basis of significant tritium transfer between these two carbons using (2S)-[2- H]tryptophan as substrate (277). This is consistent with a single active site base catalyzing a supra-facial proton transfer with respect to the putative aminoacrylate intermediates [Eq. (55)] ... 

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. 

Fig. 25. A general mechanism of /8 replacement and P elimination-deamination reactions involving a common aminoacrylate intermediate (1).

Fig. 27. A mechanism for the tryptophan synthetase, 2i82> catalysed reaction. The scheme shows that the indole formed in the reaction remains bound to the enzyme for condensation with the aminoacrylate intermediate.

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. 

Certain early speculations about the mechanism of formation of cystine from methionine contained shrewd inferences ivhich later work has shown to be correct in part. Brand and co-workers- first postulated transfer of the sulfur from a 4-carbon to a 3-carbon chain. These workers suggested that homocysteine condensed with a-aminoacrylic acid to form an intermediate compound which was cleaved to yield cysteine. Toennies suggested the direct transsulfuration between methionine... 

The unsaturated intermediate formed in this manner may dissociate off a-aminoacrylic acid, which immediately reacts spontaneously with water to form pyruvic acid and ammonia. This is the essence of the media-nism of the serine deaminase reaction. It is shown structurally in F. 3. The same mechanism applies to the deamination of other /8-hydroxyaniino acids, such as threonine, discussed in the next section. 

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