Isotopic exchange amino acids

The activation to the attack of pyrophosphate is measured by the pyrophosphate exchange technique. The enzyme, the amino acid, and ATP are incubated with [32P]-labeled pyrophosphate so that /f,y-labeled ATP is formed by the continuous recycling of the E-AA-AMP complex. The complex is formed as in equation 7.13, and the reaction is reversed by the attack of labeled pyrophosphate to generate labeled ATP. This process is repeated until the isotopic label is uniformly distributed among all the reagents. 

As with arene-amine radical ion pairs, the ion pairs formed between ketones and amines can also suffer a-deprotona-tion. When triplet benzophenone is intercepted by amino acids, the aminium cation radical can be detected at acidic pH, but only the radical formed by aminium deprotonation is detectable in base (178). In the interaction of thioxanthone with trialky lamines, the triplet quenching rate constant correlates with amine oxidation potential, implicating rate determining radical ion pair formation which can also be observed spectroscopically. That the efficiency of electron exchange controls the overall reaction efficiency is consistent with the absence of an appreciable isotope effect when t-butylamine is used as an electron donor (179). 

The various reactions undergone by coordinated amino acids have been the subject of several reviews28,31,32,438,446 and only a brief discussion will be given here. The reactions which occur can be roughly classified under three headings (a) aldol condensations, (b) reactions of complexes of amino acid Schiff bases, and (c) isotopic exchange and racemization at the a-carbon of the amino acid. 

Following structure-activity studies, the adenylate is thought to be stabilized within a cleft formed between the two subdomains of the activation domain [41,58], The rate is thus related to the formation, presence, and stability of this mixed anhydride with respect to PPi, and at high MgATP2 concentrations, with respect to ATP in the formation of diadenosine tetraphosphate (A2P4). Thus a high rate of the amino acid-dependent isotope exchange does not necessarily reflect the efficiency of adenylate formation, and certainly not the efficiency of incorporation of an amino acid into peptidyl intermediates or the final product. 

Radical enzymes are, like radical reactions in general, usually characterised by high turnover for the catalytic processes. It is hence rather difficult to study these reactions experimentally in order to gain direct insight into the mechanisms. In addition, several of the enzymes are membrane bound or anaerobic, why the determination of the crystal structures of many of these has been a formidable task. Most of the mechanistic proposals have thus been based on mutagenesis experiments (site specific exchange of an amino acid), kinetic measurements and isotope effects, and studies of inhibitor by-products . 

Figure 18.1 Isocratic separation of 17 amino acids, the early appearing ones being insufficiently resolved and the last one not eluted (reproduced by permission of A. Serban, Isotope Department, Weizmann Institute of Science, Rehovot). Conditions sample, 50 ixl containing 5 nmol of each amino acid column, 15 cm x 4 mm i.d. stationary phase. Amino Pac Na-2 (cation exchanger) 7 im mobile phase, 0.4 ml min sodium citrate 0.2 N pH 3.15-sodium phosphate 1 N pH 7.4 (1 1) temperature, BB C VIS detector 520nm after derivatization with ninhydrine.

Labeled amino acides. [DsJ-L-Tryptophan and [DsJ-tryptamin were prepared by the deuterium-exchange method as previously described. [ CsJ-L-Isoleucine and [ N2]-L-arginine were obtained from Cambridge Isotope Laboratories and Spectra Stable Isotopes, respectively. 

---