Tryptophan synthase mechanism

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... 

The tryptophan synthase bienzyme complex from enteric bacteria provides an important example wherein RSSF has been used to good advantage for the study of both enzyme mechanism and protein structure-function relationships. This enzyme complex is composed of heterologous a- and P2-subunits arranged in a nearly linear a-(3-(l-a array (81). The a-subunit catalyzes the aldolytic cleavage of IGP to indole and G3P, while the P-subunit catalyzes the PLP-dependent condensation of i-Ser and indole to yield i-Trp. The aP-reaction is essentially the sum of the individual a- and P-reactions (scheme I). Indole, the common intermediate produced at the a-site, is direcdy channeled to the P-active site via a tunnel located in the interior of the protein complex which directly interconnects the a- and P-catalytic centers (81-84). Although the individual subunits may be isolated and are functional, formation of the bienzyme complex not only increases the catalytic activities of the separate subunits by nearly 100-fold, but also alters the thermodynamic stability of P-site reaction intermediates and introduces heterotropic allosteric interactions between sites. 

In the case of tryptophan synthase, qualitative examination of the RSSF spectra has resulted in the direct detection of most of the expected reaction intermediates, and in the elucidation of the sequence of catalytic events, information crucial to the determination of the reaction mechanism. The RSSF data also provide a rational approach both for the selection of wavelengths for the detailed analysis of the dependence of relaxation rates on substrate concentrations by SWSF and for the accurate determination of isoabsorptive points by singlewavelength methods (85, 86). The presence of apparent isoabsorptive points during one or more phases of a multistep reaction simplifies gready the interpretation of physical events observed during either RSSF or SWSF rapid-kinetic studies. 

Figure 26 Mechanism of the tryptophan synthase /3-reaction. The enzyme-substrate intermediates formed in the reaction and the residues involved in catalysis are shown.

One emphasis of research in our laboratory has been the investigation of the catalytic mechanisms of NAD -requiring dehydrogenases. A second area of research has focused on PLP-dependent enzymes that catalyze either /3 or y elimination/replacement reactions.RSSF spectroscopy has been particularly useful for the characterization of the reaction mechanism, allosteric interactions, and structure-function relationships in the tryptophan synthase multienzyme complex from enteric bacteria. 

Fig. 10. Mechanism of the reactions catalyzed by both the a and B subunits of the tryptophan synthase bienzyme complex. The a reaction involves cleavage of IGP. The g reaction involves two stages stage I is reaction of L-Ser with E(Ain) to give E(A-A) stage II is reaction of indole with E(A-A) to give L-Trp. Indole, the common intermediate, is directly channeled between the two catalytic centers via a 25-A-long tunnel that interconnects the a and B active sites through the interior of the protein. See text for details.

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). 

Fig. 23. Mechanism of reaction of tryptophanase and tryptophan synthase. Reprinted from Reference 43 with permission of the American Chemical Society.

Tryptophan pool size is regulated by feedback inhibition of anthranilate synthase by tryptophan. Other mechanisms, as yet unidentified, may regulate tryptophan and indoleacetic acid synthesis. 

In . coli tryptophan synthase, which is also an heterologous 0L2P2 tetramer, conformational changes induced by the complementary subunit have received experimental support. The isolated P2 dimer exhibits only 2% of enzymatic reactivity of the a2j82 molecule (Janofsky and Crawford, 1972). Interactions between 2 and P2 subunits mutually induced conformational changes in both kind of protomers (Faeder and Hammes, 1971). Mechanism of reconstitution have been investigated by kinetic studies (Bartholmes et a/., 1980). 

Figure 1. Hypothetical mechanism for shuttling of intermediates of the common aromatic pathway between plastidic and cytosolic compartments. Enzymes denoted with an asterisk (DAHP synthase-Co, chorismate mutase-2, and cytosolic anthranilate synthase) have been demonstrated to be isozymes located in the cytosol. DAHP molecules from the cytosol are shown to be shuttled into the plastid compartment in exchange for EPSP molecules synthesized within the plastid. Abbreviations C3, phosphoenolpyruvate C4, erythrose 4-P DAHP, 3-deoxy-D-arabino-heptulosonate 7-phosphate EPSP, 5-enolpyruvylshikimate 3-phosphate CHA, chorismate ANT, anthranilate TRP, L-tryptophan PPA, prephenate AGN, L-arogenate TYR, L-tyrosine and PHE, L-phenylalanine.

Behavioral disorders such as anorexia, sleep disturbances, and pain insensitivity associated with hyperammonemia have been attributed to increased tryptophan transport across the blood-brain barrier and the accumulation of its metabolites. Two of the tryptophan-derived metabolites are serotonin and quinolinic acid (discussed later). The latter is an excitotoxin at the N-methyl-D-aspartate (NMDA) glutamate receptors. Thus, the mechanism of the ammonium-induced neurological abnormalities is multifactorial. Normally only small amounts of NH3 (i.e., NH4 ) are present in plasma, since NH3 is rapidly removed by reactions in tissues of glutamate dehydrogenase, glutamine synthase, and urea formation. 

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. 

To rationalize these data, Floss et al. proposed the mechanism shown in Scheme 55 wherein a direct electrophilic aromatic substitution reaction from an enzyme-bound DMAPP ion pair species alkylates C-4 of the tryptophan nucleus [82]. The minor product, where partial loss of stereochemical integrity is sacrificed, was envisioned to occur via rotation around the C-l/C-2 bond of the allylic carbocation species as shown in Scheme 55. Poulter et al. subsequently published a mechanistic study on DMAT synthase that is fully consistent with this interpretation [84]. 

Biosynthesis of some classes of terpene indole alkaloids is well understood. In certain cases, many of the enzymes that are responsible for biosynthesis have been cloned and mechanistically studied. In other cases, biosynthesis pathway is only proposed based on the results of feeding studies with isotopically labeled substtates and from the structures of isolated biosynthetic intermediates. All terpene indole alkaloids are derived from tryptophan and the iridoid terpene secologanin (Fig. 14.11). Tryptophan decarboxylase, a pyridoxal-dependent enzyme [29], converts tryptophan to tryptamine [30]. The following strictosidine synthase-catalyzed Mannich reaction connects ttyptamine and secologanin to yield strictosidine [31]. The Apocynaceae, Loganiaceae, Rubiaceae, and Nyssaceae families of plants each produce terpene indole alkaloids with dramatically diverse structures [32-34]. The mechanisms and control of... 

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