Amino Tryptophan synthetase

Most of the proteins on which fragment folding studies have been carried out are extracellular. Of the nine discussed in this article, only tryptophan synthetase and /3-galactosidase are not secreted. Many secreted proteins are synthesized with 20 or so additional amino acid residues at the N-terminus of the peptide chain (Blobel... 

Schneider, W. P., Nichols, B. P., and Yanofsky, C. (1981). Procedure for production of hybrid genes and proteins and its use in assessing significance of amino acid differences in homologous tryptophan synthetase alpha polypeptides. Proc. Natl. Acad. Set. USA, 78, 2169-2174. 

Transamination is just one of a wide range of amino acid transformations that are catalyzed by PLP enzymes. The other reactions catalyzed by PLP enzymes at the a-carbon atom of amino acids are decarboxylations, deam-inations, racemizations, and aldol cleavages (Figure 23.12). In addition, PLP enzymes catalyze elimination and replacement reactions at the P-carbon atom (e.g., tryptophan synthetase Section 24.2.11) and the y-carbon atom (e.g., cytathionine P-synthase, Section 24.2.9) of amino acid substrates. Three common features of PLP catalysis underlie these diverse reactions. 

TMany of the PLP enzymes that catalyze amino acid transformations, such as serine hydroxymethyltransferase, have a similar structure and are clearly related by divergent evolution. Others, such as tryptophan synthetase, have quite different overall structures. Nonetheless, the active sites of these enzymes are remarkably similar to that of aspartate aminotraas-ferase, revealing the effects of convergent evolution. 

In the presence of both the substrates, indole-3-glycerolphosphate and L-serine, tryptophan synthetase, 02)82 catalyses two parallel reactions. The a subunits in the complex carry out the cleavage of indole-3-glycerolphosphate releasing glyceralde-hyde-3-phosphate in the medium but the indole remains bound to the complex. Concomitantly, the /Sj subunits promote the conversion of serine into the aminoacrylate-pyridoxal-P species (Fig. 27, 1) which acts as a Michael acceptor for the indole producing the L-tryptophan-pyridoxal-P complex from which the amino acid is released by hydrolysis (Fig. 27). That the aforementioned molecular events can be described in such vivid detail is due to an ingenious series of partial reactions... 

Proteins associate with each other to form quaternary structures. Many proteins consist of more than one subunit. For example, hemoglobin has a molecular weight of 64,000 and is composed of four subunits, each of molecular weight 16,000. Two of the subunits are alike, and two are different. The enzyme tryptophan synthetase from Escherichia coli, which catalyzes the final two steps in the biosynthesis of that amino acid, consists of two nonidentical subunits, each of which catalyzes one reaction. Other enzymes contain regulatory and catalytic subunits. Still other enzymes consist of aggregates of two, three, or more identical subunits. The specific, noncovalent association of protein subunits is termed the quaternary stmcture of a protein. If the subunits are not identical, the association is called heterotypic. The association of identical subunits is termed homotypic. 

Auxotrophs of E. coli elaborate the enzyme tryptophan synthetase. The enzyme molecule is composed of two different polypeptides, referred to as polypeptides A and B. Polypeptide A has been extensively purified and its molecular weight is known to be 26,000. Although the full amino acid sequence of polypeptide A is not yet known, it is possible to digest the molecule, separate smaller polypeptidic fragments, and establish the sequence of the small polypeptide fragments. One of these polypeptides, referred to as peptide CP2, has the following sequence ... 

Inhibitors of this type have been used in the irreversible inhibition of pyridoxal-linked aspartate aminotransferase, y-cystathionase, and tryptophan synthetase. These inhibitors are y-unsaturated amino acids. Aspartate aminotransferase is inhibited by molecules 1 and 2,... 

Yanofsky, C., Drapeau, G. R., Guest, J. R. and Carlton, B. C. (1967) The complete amino acid sequence of the tryptophan synthetase A protein (a-subunit) and its colinear relationship with the genetic map of the A gene. Proc. Nat. Acad. Sci. 57, 296-298. 

Murgola, E. j. and Yanofsky, C. (1974) Selection for new amino acids at position 211 of the tryptophan synthetase a-chain of Escherichia coli. J. Molec. Biol. 86, 775-784. 

Tryptophan synthetase has been shown to be a pyridoxal phosphate-requiring enzyme 285, 287). Tatum and Shemin 286) studied the mechanism of the reaction by labeling the serine with deuterium and N. The was retained in the tryptophan, showing that the amino group of serine was not removed in the course of the condensation. The experiments with deuterium indicated that the a-hydrogen of serine was removed in the reaction. 

Another reason for concluding that peptide antibiotics are not synthesized by the kind of template mechanism that is operative in protein formation is that variation of amino acids in the environment can shift the proportion of the molecular species of peptides that are being produced. In protein synthesis, specificity of amino acid sequence is under direct genetic control and single amino acid substitutions in such proteins as hemoglobin, tryptophane synthetase, or phage coat proteins are attributed to alteration in the nucleotide sequence of structural genes rather than to environmental variation (Mach and Tatum, 1964). 

Applications of tryptophan synthetase Tryptophan synthetase (EC 4.2.1.20) is a pyridoxal phosphate-dependent enzyme that, in the cell, catalyzes the a,/3-elimination of water from serine to form a pyridoxyl-bound a-aminoacrylate, which undergoes Michael addition of indole to form the named amino acid. This type of reaction has been used to prepare (5)-tryptophan isotopomers with a variety of labeling patterns by use of different labeled indoles and (5)-serines in yields of up to 98% based on indole and 92% based on (5)-serine. 

The enzyme s ability to form this Michael acceptor intermediate has been exploited for the preparation of other labeled amino acids by using alternative nucleophiles. For example, tryptophan synthetase, isolated from a strain of E. coli engineered to overexpress this enzyme, was applied to the synthesis of several carbon-13 and deuterium isotopomers of 5-benzyl-(5)-cysteine from incubations of the corresponding labeled (5)-serines in the presence of benzyl mercaptan as the nucleophile instead of indole. The resulting... 

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. 

The information contained in the DNA (i.e., the order of the nucleotides) is first transcribed into RNA. The messenger RNA thus formed interacts with the amino-acid-charged tRNA molecules at specific cell organelles, the ribosomes. The loading of the tRNA with the necessary amino acids is carried out with the help of aminoacyl-tRNA synthetases (see Sect. 5.3.2). Each separate amino acid has its own tRNA species, i.e., there must be at least 20 different tRNA molecules in the cells. The tRNAs contain a nucleotide triplet (the anticodon), which interacts with the codon of the mRNA in a Watson-Crick manner. It is clear from the genetic code that the different amino acids have different numbers of codons thus, serine, leucine and arginine each have 6 codewords, while methionine and tryptophan are defined by only one single nucleotide triplet. 

Discrimination between some pairs of tRNAs depends entirely on the anticodon sequence. For example, tRNAMet contains the anticodon CAU. That for a minor tRNAIle is the same except that the cytosine has been posttranscriptionally modified by covalent linkage of a molecule of lysine via its e-amino group to C2 of the cytosine. The latter base (Iysidine) is correctly recognized by E. coli isoleucyl-tRNA synthetase but, if the cytosine is unmodified, it is aminoacylated by methionyl-tRNA synthetase.192 In most instances the acceptor specificity, or tRNA identity, is not determined solely by the anticodon sequence. Thus, when a methionine initiator tRNA was modified to contain a tryptophan anticodon, it was only partially charged with tryptophan in vivo. However, when A73 of the methionine tRNA was also converted to G73, only tryptophan was inserted.193 Nucleotide 73 (Fig. 

As any elementary textbook on molecular biology will relate, the sequences of proteins are stored in DNA in the form of a triplet code. Each amino acid is encoded by one or more triplet combinations of the four bases A, T, G, and C. For example, tryptophan is coded by the sequence TGG. The sequence of triplets is converted into a protein by a process in which DNA is first transcribed into mRNA. This message is then translated into protein on the ribosomes in conjunction with tRNA and the aminoacyl-tRNA synthetases. In prokaryotes, there is a one-to-one relationship between the sequence of triplets in the DNA. and the sequence of amino acids in the protein. In eukaryotes, the DNA often contains stretches of intervening sequences or introns which are excised from the mRNA after transcription (Chapter 1). 

---