Tryptophan-activating enzyme

The molecular interaction between the activating enzyme and the amino acid is not known, but it probably varies with the type of activating enzyme. Some enzymes (. g., the alanine-activating enzyme) are unaffected by paramercuric benzoate, but others, like the tryptophan-activating enzyme, appear to be SH enzymes the activity of which depends on the presence of free SH groups in the molecule. Potassium ions are known to activate the tyrosine enzyme. 

A transfer of the adenylyl group i.e., a P—0 split has not been obtained so far (or maybe has not been looked for) with the adenylates in the absence of enzyme. In the presence of the activating enzymes, on the other hand, the adenylyl group is veiy readily transferred onto pyrophosphate to give ATP 116, 169, 164, 177). This reaction, however, is not specific, since tryptophan activating enzyme, for example, can synthetize ATP from all the amino acid adenylates tested 116) as well as from the adenylate of D-tiyptophan 169). The same transfer of the AMP moiety onto PP does, of course, also occur in the amino acid catalyzed ATP-PP exchange reaction. 

The existence of an acyl-adenylate bound to enzyme has been demonstrated in the case of tryptophan activation (344). Tryptophan was incubated with ATP in the presence of substrate quantities of purified tryptophan-activating enzyme. Adenyl-tryptophan was isolated and chemically characterized after its removal from the denatured enzyme. 

Isolation of alkaline phosphatase from Escherichia coli in which 85% of the proline residues were replaced by 3,4-dehydro-proline affected the heat lability and ultraviolet spectrum of the protein but the important criteria of catalytic function such as the and were unaltered (12). Massive replacement of methionine by selenomethionine in the 0-galactosidase of E. coli also failed to influence the catalytic activity. Canavanine facilely replaced arginine in the alkaline phosphatase of this bacterium at least 13 and perhaps 20 to 22 arginyl residues were substituted. This replacement by canavanine caused subunit accumulation since the altered subunits did not dimerize to yield the active enzyme (21). Nevertheless, these workers stated "There was also formed, however, a significant amount of enzymatically active protein in which most arginine residues had been replaced by canavanine." An earlier study in which either 7-azatryptophan or tryptazan replaced tryptophan resulted in active protein comparable to the native enzyme (14). 

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

According to the authors tetrahydrobiopterin has a range of co-factor roles, including being required for the activity of tyrosine and tryptophan hydroxylase, enzymes that are essential for dopamine and serotonin synthesis. They speculated that something present in the heroin pyrolysate, a product formed when heroin is heated to 250° C, inhaled by the patient, acted as a reversible inhibitor of tetrahydrobiopterin metabolism, providing a biochemical explanation for impairment of dopamine metabolism and Parkinsonism in this case. 

CD spectroscopy has also provided valuable insight into the chemical stability and chemical denaturation of proteins. A recent study by Rumfeldt etal. examines the guanidinium-chloride induced denaturation of mutant copper-zinc superoxide dismutases (SODs). These mutant forms of the Cu, Zn-SOD enzyme are associated with toxic protein aggregation responsible for the pathology of amyotrophic lateral sclerosis. In this study, CD spectroscopy was used in conjunction with tryptophan fluorescence, enzyme activity, and sedimentation experiments to study the mechanism by which the mutated enzyme undergoes chemical denaturation. The authors found that the mutations in the enzyme structure increased the susceptibihty of the enzyme to form partially unfolded destabilized monomers, rather than the stable metaUated monomer intermediate or native metallated dimer. 

The effect of proteins on pollutant toxicity includes both quantitative and qualitative aspects. Experiments show that animals fed proteins of low biological value exhibited a lowered microsomal oxidase activity when dietary proteins were supplemented with tryptophan, the enzyme activity was enhanced. Alteration of xenobiotic metabolism by protein deprivation may lead to enhanced or decreased toxicity, depending on whether metabolites are more or less toxic than the parent compound. For example, rats fed a protein-deficient diet show decreased metabolism but increased mortality with respect to pentobarbital, parathion, malathion, DDT, and toxaphene (Table 6.4). On the other hand, rats treated under the same conditions may show a decreased mortality with respect to heptachlor, CC14, and aflatoxin. It is known that, in the liver, heptachlor is metabolized to epoxide, which is more toxic than heptachlor itself, while CC14 is metabolized to CC13, a highly reactive free radical. As for aflatoxin, the decreased mortality is due to reduced binding of its metabolites to DNA. 

Reaction Mechanism. The following reaction mechanism is compatible with the above data. Tryptophan first combines with ferrous enzyme and activates the heme in the enzyme. The activated enzyme then reacts with oxygen to form an intermediary ternary complex. Both substrates, tryptophan and oxygen, are activated in the complex and interact, yielding formylkynurenine as product. 

There is currently some uncertainty about the complexity of the tryptophan gene-enzyme relationships in B. subtilis. Whitt and Carlton [46,47] have noted pleiotropic effects. Most recently [46] they have found that the pleiotropy is limited to elimination of InGPS activity by mutations in either the trpD or trpF genes, which are primarily concerned with PRT and PRAI activity, respectively. The pleiotropic effects are shown in Fig. 3 by dotted lines and may indicate that the enzymes function as aggregates in vivo or may represent effects on translation similar to polarity effects. Hoch et al. [48], on the other hand, have not found these pleiotropic effects and report essentially one gene-one enzyme activity [when the individual activities of the tryptophan synthetase a and p2 subunits (Fig. 1) are included]. The nonconformity in the results of different investigators may be due to the use of different mutants and to different methods of preparation, affecting enzyme stabilities. 

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