Amidotransferases

Liver, the major site of purine nucleotide biosynthesis, provides purines and purine nucleosides for salvage and utilization by tissues incapable of their biosynthesis. For example, human brain has a low level of PRPP amidotransferase (reaction 2, Figure 34-2) and hence depends in part on exogenous purines. Erythrocytes and polymorphonuclear leukocytes cannot synthesize 5-phosphoribosylamine (strucmre III, Figure 34-2)... 

Hepatic purine nucleotide biosynthesis is stringently regulated by the pool size of PRPP and by feedback inhibition of PRPP-glutamyl amidotransferase by AMP and GMP. 

Table 4.1 Catalytic properties and inhibition by glutamyl-y-boronate of the glutaminase activity of bacterial Glu-tRNAGln amidotransferase... 

Figure 2 Indirect pathway for Asn-tRNA " and Gln-tRNA° " biosynthesis. ND-AspRS, nondiscriminating aspartyl-tRNA synthetase ND-GluRS, nondiscriminating glutamyl-tRNA systhetase AdT, aminoacyl-tRNA amidotransferase.

Figure 3 Reaction mechanism of aminoacyl-tRNA amidotransferase. For Asp-tRNA " amidotransferase (AspAdT), n =, tRNA = tRNA =" for Glu-tRNA° " amidotransferase (GiuAdT), n = 2, tRNA =tRNA° ".

Figure 5 Structure of the archaeai GatDE aminoacyi-tRNA amidotransferase. GatDE forms a heterotetramer, with two GatDE proteins binding through their GatD subunits. Binding of tRNA° " induces a conformational shift of the helical and tail domains, which scan the D-ioop and the T-arm. This figure was reproduced from H. Oshikane K. Sheppard S. Fukai ...

Figure 7 The direct and indirect pathways of tRNA asparaginylation. The direct pathway consists of charging by AsnRS on tRNA " of free Asn formed with asparagine synthetase A or B. The Asn-tRNA " binds the EF-Tu factor in bacteria (or EF-1A in eukaryotes and archaea) to be carried to the ribosome, in the indirect pathway, a nondiscriminating AspRS (ND-AspRS) charges Asp on tRNA " Asp-tRNA " does not bind the eiongation factor but is converted by the tRNA-dependent trimeric amidotransferase GatCAB into Asn-tRNA ", which binds the EF-Tu factor and is carried to the ribosome where it is used for polypeptide chain elongation.

Figure 10 The transamidosome of Thermus thermophilus catalyzing tRNA asparaginylation. (a) Formation of the transamidosome (I) The AspRS (in blue) binds tRNA " (in gray, Kq = 2 pmol l ) before association of the amidotransferase GatCAB (in orange, Kq = 0.6 pmol l ) to form the ternary complex, (ii) The free GatCAB binds tRNA " with a poor affinity (Kd > 10 pmol r ) before association of tRNA " thus pathway (i) is preferred forthe formation of the transamidosome. (b) The catalytic cycle of the transamidosome. In the absence of free tRNA ", the transamidosome aminoacylates tRNA " with a first-order rate constant of 0.017 s (1) and amidates the tRNA "-bound Asp into Asn with a rate constant of 0.11 s (2). In the presence of an excess of free tRNA ", the first Asn-tRNA " is formed with a rate constant of 0.094 s (3), whereas the following catalytic cycles occur with a rate constant of 0.043 s (4), indicating that dissociation of the newly formed Asn-tRNA " accompanied by the disruption of the complex is rate-limiting at the steady state.

Indirect Pathway Inhibitors of Glutamyl-tRNA Synthetase, Aspartyl-tRNA Synthetase, and Aminoacyl-tRNA Amidotransferase... 

Table 5 Inhibitors of Helicobacter pylori GatCAB amidotransferase derived from puromycin...

GatCAB amidotransferase.This natural product mimics the charged 3 -terminus of aa-tRNA and has been used as a tool for the study of protein biosynthesis. The parent compound 22 is a very weak inhibitor of AdT. The amino acid chain is related to tyrosine and differs from the glutamic and aspartic side chains transformed in the kinase or the transamidase steps. Replacement of the methoxyphenyl moiety of puromycin by carboxylic acid derivatives (23-26) improved the ability to inhibit this AdT. Stable analogues of the transition state in the last step of the transamidation process (27-29) where the carbonyl to be attacked by NH3 is replaced by tetrahedral sulfur or phosphorus atom with a methyl group mimicking ammonia exhibited the highest activity. 

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