Tryptophan synthase studies

Tryptophan synthase (EC 4.2.1.20) from bacteria is a classic multienzyme complex that channels a metabolic intermediate (for reviews and commentaries see 1-6)). Structure/ function analysis of tryptophan synthase was pioneered by genetic and biochemical investigations beginning in the mid-1940 s. This chapter emphasizes the relationship between the function of tryptophan synthase and the three-dimensional structure of the tryptophan synthase 02)82 complex from Salmonella typhimurium7,8) and focuses on studies carried out since a recent review.6 ... 

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 ... 

Our laboratory has studied the stereochemistry of methyl group formation in a number of a, 0 elimination reactions of amino acids catalyzed by pyridoxal phosphate enzymes. The reactions include the conversions of L-serine to pyruvate with tryptophan synthase 02 protein (78) and tryptophanase (79), of L-serine and l-tyrosine with tyrosine phenol-lyase (80), and l-cystine with S-alkylcysteine lyase (81). In the latter study, the stereospecific isotopically labeled L-cystines were obtained enzymatically by incubation of L-serines appropriately labeled in the 3-position with the enzyme O-acetyl serine sulfhy-drase (82). The serines tritiated in the 3-position were prepared enzymatically starting from [l-3H]glucose and [l-3H]mannose by a sequence of reactions of known stereochemistry (81). The cysteines were then incubated with 5-alkyl-cysteine lyase in 2H20 as outlined in Scheme 19. The pyruvate was trapped as lactate, which was oxidized with K2Cr202 to acetate for analysis. Similarly, Cheung and Walsh (71) examined the conversion of D-serine to pyruvate with... 

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. 

This enzyme represents an interesting contrast to tryptophan synthase, which catalyzes the essentially irreversible formation of i-Trp. The spectrum of the native enzyme, which is highly pH dependent, is characterized by two absorbance bands centered at 420 nm and 337 nm. Early RSSF investigations utilizing rapid incremental jumps in pH showed that the two spectral bands arise from different protonation states of the covalently bound internal aldimine, E(Ain), form of the cofactor (101). Studies with a variety of amino acid inhibitors of tryptophanase (amino acids, which react reversibly with the enzyme to form covalent PLP-intermediates, but cannot complete the P-elimination reaction to form products), showed that the 420-nm species is the reactive form of the cofactor. The 337-nm species must be converted to the 420-nm species before reaction with the amino group of the substrate will occur. The 420-nm species represents aketoenamine form of the cofactor in which the iminium nitrogen of the Schiff s base is protonated (102). 

The presence of a suitable chromophore, such as the PLP cofactor, makes it possible to investigate protein structure-function relationships that may be far removed from the chromophoric site. Tryptophan synthase is considered to be a prototype multienzyme complex in which metabolites are channeled directly between successive metabolic enzymes. As described above, allosteric interactions serve to coordinate catalytic events between the heterologous active sites in the complex. Such close interactions suggest that mutations in one enzyme may affect the reactivity of the other. We have found it possible to study the consequences of mutations in the a-subunit by looking for changes in the presteady state behavior of reactions catalyzed at the (3-site (88, 89). Since amino acid replacements in the a-subunit will not affect the primary amino acid sequence of the P-subunit, alterations in the reactivity of the a2P2 complex will be due primarily to differences in the reactivity of the a-subunit and/or aP-subunit interactions. 

The stereochemistry of the PLP-dependent elimination/substitution reactions involving the /3- and -y-carbons of amino acid substrates generally conforms to the trends observed among the PLP-dependent transamination and decarboxylation reactions at the a-carbon [Eq. (44)]. All of the enzymes so far studied that catalyze nucleophilic replacement reactions at the j8-carbon (five examples e.g., tryptophan synthase) and/or a,j3-elimination reactions (seven examples e.g., tryptophanase) involve retention of configuration at the jS-carbon (231) (Eq. (54)] ... 

Although the studies of the rll region of the T4 chromosome established that genetic mapping could be carried to the level of individual nucleotides in the DNA, it was still necessary to prove a linear correspondence between the nucleotide sequence in the DNA and the amino acid sequence in proteins. This was accomplished by Yanofsky - and associates through study of the enzyme tryptophan synthase of... 

The enzyme cysteine synthase (EC 4.2.99.8) catalyzes the last step in the biosynthesis of cysteine, converting serine acetate 97 to cysteine 134. Floss et al. (84) studied this -replacement reaction using stereospecifically tritiated serine acetates prepared from the labeled serines 60 synthesized as in Scheme 18 (Section IV). Assessment of chirality of the cysteine produced by degradation to serine and use of tryptophan synthase showed that the j3-replacement reaction 97a134 had proceeded with retention of configuration (84) (Scheme 45),... 

Bilsel, O., Yang, L., Zitzewitz, J. A., Beechem, J. M. and Matthews, C. R. 1999, Time-Resolved Fluorescence Anisotropy Study of the Refolding Reaction of the a-Subunit of Tryptophan Synthase Reveals Nonmonotonic Behavior of the Rotational Correlation Time. Biochemistry 38, 4177 - 4187. 

So far, none of the enzymes leading to tryptophan after AS (152,158) has been studied in C. roseus. Only a time course of tryptophan synthase... 

To illustrate the use of RSSF spectroscopy to study enzyme catalysis, we give two systems detailed treatment herein, namely, horse liver alcohol dehydrogenase and Salmonella typhimurium tryptophan synthase. For an inclusive review of RSSF spectroscopy applications in the field of enzymology, see Brzovid and Dunn. ... 

Molecular modelling using spheres has also been used to study complexes formed from different subunits, i.e. tryptophan synthase (0 2/82) and its a and 2 subunits [122], and likewise the DNA-dependent RNA polymerase (y8 /8 2a) and its a,... 

The fifth and terminal step of tryptophan synthesis, the removal of the glycerolphosphate side chain and its replacement by the alanyl moiety of L-serine [Fig. 4 (17)], is catalyzed by the structurally complex enzyme tryptophan synthase (TS). This protein, probably the earliest known multicomponent enzyme, has been extensively studied and often reviewed (Crawford, 1975). The complex nature of the enzyme is illustrated by its capability of catalyzing the following distinct reactions ... 

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