Tryptophan in enzymes

Apart from the valuable data about the involvement of tryptophan in enzymic processes (Section VII.), which can be obtained by use of sulfenyl halides, the analytical utility of this reaction is another major application. By reaction with nitrophenylsulfenyl halides a chromophore is generated in a protein which absorbs in the visible part of the spectrum. This permits easy quantitation of the reaction, and thereby allows determination of the tryptophan content of a protein (see Section IV.4.2. for the analytical application of the reaction). 

Spande, T. F., N. M. Green, and B. Witkop The Reactivity toward N-bromo-succinimide of Tryptophan in Enzymes, Zymogens, and Inhibited Enzymes. Biochemistry 5, 1926-1933 (1966). 

Figure 4.6 The bifunctional enzyme PRA-isomerase (PRAI) IGP-synthase (IGPS) catalyzes two sequential reactions in the biosynthesis of tryptophan. In the first reaction (top half), which is catalyzed by the C-terminal PRAI domain of the enzyme, the substrate N-(5 -phosphoribosyl) anthranilate (PRA) is converted to l-(o-carboxyphenylamino)-l-deoxyribulose 5-phosphate (CdRP) by a rearrangement reaction. The succeeding step (bottom half), a ring closure reaction from CdRP to indole-3-glycerol phosphate (IGP), is catalyzed by the N-terminal IGPS domain.

Figure 4.7 Two of the enzymatic activities involved in the biosynthesis of tryptophan in E. coli, phosphoribosyl anthranilate (PRA) isomerase and indoleglycerol phosphate (IGP) synthase, are performed by two separate domains in the polypeptide chain of a bifunctional enzyme. Both these domains are a/p-barrel structures, oriented such that their active sites are on opposite sides of the molecule. The two catalytic reactions are therefore independent of each other. The diagram shows the IGP-synthase domain (residues 48-254) with dark colors and the PRA-isomerase domain with light colors. The a helices are sequentially labeled a-h in both barrel domains. Residue 255 (arrow) is the first residue of the second domain. (Adapted from J.P. Priestle et al., Proc.

The trp repressor controls the operon for the synthesis of L-tryptophan in Escherichia coli by a simple negative feedback loop. In the absence of L-tryptophan, the repressor is inactive, the operon is switched on and the enzymes which synthesize L-tryptophan are produced. As the concentration of L-tryptophan increases, it binds to the repressor and converts it to an active form so that it can bind to the operator region and switch off the gene. 

This similarity between MDMA and PCA is also observed in vivo in that PCA produces both an acute and long-term depletion of 5-HT (Fuller et al. 1975 Steranka et al. 1977). Like PCA, the acute decrease in 5-HT concentrations produced by MDMA is associated with a decrease in the activity of the rate-limiting enzyme for 5-HT synthesis, tryptophan hydroxylase (TPH). The timecourse of this change in cortical enzyme activity is also shown in figure 1. More detailed analysis of this acute effect of MDMA and kinetic analysis of TPH activity reveals that the decrease in enzyme activity actually precedes the decline in transmitter levels and is due to a reduction in the activity of the enzyme (Schmidt and Taylor 1987 Schmidt and Taylor 1988). As shown for the cortex in figure 3, the decrease in 5-HT... 

Serotonergic neurons contain the enzyme L-tryptophan-5-monooxygenase (EC 1.14.16.4), more commonly termed tryptophan hydroxylase, which converts tryptophan to 5-hydroxytryptophan (5-HTP) (Fig. 13-5). Tryptophan hydroxylase contains 444 amino acids, corresponding to a molecular weight of about 51 Da. This enzyme is synthesized in serotonergic cell bodies of the raphe nuclei and is found only in cells that synthesize 5-HT. Therefore its distribution in brain is similar to that of 5-HT itself. The Km of tryptophan hydroxylase for tryptophan is approximately 30-60 pmol/1, a concentration comparable to that of tryptophan in brain. If the concentration of tryptophan in serotonergic neurons is assumed to be comparable to that in whole brain, the enzyme would not be saturated with substrate, and the formation of 5-HT in brain would be expected to rise as the brain concentration of tryptophan increases. This has been found to occur in response to raising the dietary intake of tryptophan specifically. 

Vitamin Ba (pyridoxine, pyridoxal, pyridoxamine) like nicotinic acid is a pyridine derivative. Its phosphorylated form is the coenzyme in enzymes that decarboxylate amino acids, e.g., tyrosine, arginine, glycine, glutamic acid, and dihydroxyphenylalanine. Vitamin B participates as coenzyme in various transaminations. It also functions in the conversion of tryptophan to nicotinic acid and amide. It is generally concerned with protein metabolism, e.g., the vitamin B8 requirement is increased in rats during increased protein intake. Vitamin B6 is also involved in the formation of unsaturated fatty acids. 

The long-lived phosphorescence of the tryptophan in alkaline phosphatase is unusual. Horie and Vanderkooi examined whether its phosphorescence could be detected in E. coli strains which are rich in alkaline phosphatase.(89) They observed phosphorescence at 20°C with a lifetime of 1.3 s, which is comparable to the lifetime of purified alkaline phosphatase (1.4 s). Long-lived luminescence was not observed from strains deficient in alkaline phosphatase. The temperature dependence of tryptophan phosphorescence in the living cells was slightly different from that for the purified enzyme, indicating an environmental effect. 

A previous study of the reaction of ozone with lysoz3mie dissolved in anhydrous formic acid gave rise to the conclusion that the only amino acid residues affected in the early stages of the reaction were the tryptophan residues 108 and 111 ( ). Conversion of these residues to N -formyl-kynurenine did not cause loss in enzyme activity. Imoto et al. (7) have pointed out that this result is anomalous since modifications of tryptophan 108 (e.g. with iodine) normally causes inactivation. 

It is an indole ethylamine formed in biological systems from the amino acid L-tryptophan by hydroxylation with tryptophan hydroxylase enzyme, followed by the decarboxylation by the nonspecific aromatic L-amino acid decarboxylase. 5-HT is then taken up into secretory granules and stored. 

In no case is the amino acid composition of a sialidase yet known, and complete insight into the amino acids involved in enzyme catalysis is unavailable. There is one report demonstrating a possible involvement of tryptophan residues in enzyme catalysis.331 Establishing of the complete, amino acid sequence of sialidases from different sub-types of influenza virus is to be expected from the determination of the nucleotide sequence of the viral genome by using a plasmid technique.332 Preliminary results revealed the identity of the first 12 amino... 

GABA analogs are used in the treatment of epilepsy and hypertension. Levels of GABA can also be increased by administering inhibitors of the GABA-degrading enzyme GABA aminotransferase. Another important neuro-transmitter, serotonin, is derived from tryptophan in a two-step pathway. 

Most bacteria and fungi have three isozymes of DAHP synthase, each controlled by feedback inhibition by one of the three products tyrosine, phenylalanine, or tryptophan. In E. coli these are encoded by genes aro F, am G, and aro H, respectively.11-123 All of the enzymes contain one atom of iron per molecule and show spectral similarities to hemerythrin.13... 

Distribution of Tryptophan Biosynthetic Enzyme Activities on Different Proteins in Bacteria and Fungi... 

The first two steps in the biosynthesis of tryptophan in Salmonella typhimurium involve the enzyme complex anthranilate synthase-phosphoribosyltransferase, which is a tetramer having two subunits of each enzyme. The anthranilate synthase catalyzes reaction (7) and the phos-phoribosyltransferase catalyzes two reactions the N-terminal portion cleaves glutamine to glutamate giving NH3 for the anthranilate synthase, while the C-terminal portion catalyzes reaction (8).3,1,312 All these reactions require M2+ cations. Orotate phosphoribosyltransferase binds four Mn2+ ions in a cooperative fashion kinetic data have been interpreted in a scheme where both metal-free and metal-containing enzyme catalyze the reaction.313... 

The production of serotonin requires the absorption of the amino acid tryptophan from your food. Transport of this amino acid is influenced by the level of other amino acids in your blood that level, in turn, is also influenced by what you eat. Within the neurons of your brain, tryptophan is converted to 5-hydroxy-tryptophan by tryptophan hydroxylase, an enzyme that is usually not saturated with substrate. Therefore, if you eat less tryptophan, your brain generally produces less serotonin. Conversely, providing additional tryptophan in the diet may lead to increased production of serotonin within neurons. It is worth noting, however, that simply producing more of any neurotransmitter does not guarantee that the neuron will actually release it. If too much serotonin is produced, then the excess is simply discarded. Studies have shown that only extreme depletion or supplementation of this amino acid in the diet can influence serotonin-controlled brain processes such as mood and sleep. 

In the biosynthesis pathways of histidine and tryptophan, the enzymes HisA (N -[(5 -phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerase) and TrpF (PRAI,phosphoribosylanthranilate isomerase) (Figure 16.13), both of which are ()3a)8-barrels, both catalyze Amadori rearrangements of a ther-molabile aminoaldose into the corresponding aminoketose (Figure 16.13). 

The acid carboxypeptidase from A. saitoi releases the carboxyterminal phenenylalanine-amide (-Phe-NH2) from the carboxy-terminal amidated peptides, such as gastrin-related peptide (/-amyloxycarbonyl (Aoc)-Trp-Met-Asp-Phe-NH2, Aoc-WMDF-NH2) and molluscan cardioexcitatoiy neuropeptide (Phe-Met-Arg-Phe-NH2, FMRF-NH2) [86], The summarized data are shown in Table 12. When gastrin-related peptide was used as a substrate, the enzyme acted only as a carboxyamidase, because of the presence of the hydrophobic amino acid residue, tryptophan, in the P3 [12] position. 

CGTases (EC 2.4.1.19) are bacterial enzymes that facilitate the biosynthesis of cyclodextrins from starch through intramolecular transglucosylation. The primary structures of most of these enzymes have been published, and the three-dimensional structure of Bacillus circulans CGTase has been established. Studies of transglucosylation molecular mechanism have indicated that amino acids such as histidine and tryptophan are implicated in such mechanisms. Nitration of CGTase with TNM induces a loss of enzyme activity, a decrease in enzyme affinity towards the (i-CD copolymer, and a loss of tryptophan fluorescence (Villette etal. 1993). 

The bacterial ferredoxins, in general, are similar to each other in amino acid content, but differ in detail with each ferredoxin having a characteristic composition. Each contains about fifty total amino acid residues with an abundance of acidic and a paucity of basic amino acids. The abundance of acidic residues accounts for the affinity of ferredoxin for DEAE-cellulose and its low isoelectric point (Lovenberg, Buchanan, and Rabinowitz (65)). Each of the bacterial ferredoxins lacks histidine, methionine, tryptophan, and at least one additional amino acid, which is characteristic of a particular ferredoxin. The possible significance of these differences in either the conformation or function of these proteins has not been established, although minor differences in enzymic activity do exist (Lovenberg, Buchanan, and Rabinowitz (65)). 

In some enzymes, the protein radical appears to participate in substrate oxidation. Evidence exists for the involvement of a surface tryptophan in the oxidation of veratryl alcohol by the ligninase from Phanerochaete chrysosporium [33]. Similarly, tryptophan radicals on the surface of the versatile peroxidases from Pleurotus eryngii and Bjerkandera adjusta [34—36], and a tyrosine in the LiP from Trametes cervina [33], are thought to be involved in substrate oxidation. 

The catabolism of d- and of L-tryptophan in P. aureofaciens is different.67 Only the latter isomer is catabolized by the kynurenine pathway, and it also induces the enzymes of this pathway. Added L-tryptophan may then be catabolized by this pathway. That which is will not be available for biosynthesis of pyrrolnitrin. In support, a mutant that lacks the first enzyme of this catabolic pathway showed a 30-fold increase in the production of pyrrolnitrin as compared to normal organisms.67 D-Tryptophan, not suffering catabolism in this way, will be more readily available, through slow conversion into L-tryptophan, for biosynthesis of... 

With ferryl myoglobin, in contrast to peroxidases, the reactions of the protein free radicals and that of the ferryl haem can be considered as uncoupled from each other. The protein has not been designed to form a cation radical for a specific reaction therefore not only is more than one cation free radical generated, but there is no control over their subsequent reactions. A similar situation can be observed in cytochrome c peroxidase mutants that have lost tryptophan-191. A different amino-acid free radical is still formed that is less stable. Indeed, even in the presence of tryptophan-191, small amounts of other free radicals are formed [237] this is further evidence that even in enzymes it is difficult to exclusively control free radical reactions. 

---