Section 7
Chapter 6,570

Synthesis of modified tryptophanyl adenylates and modified atp and their use as tools for elucidation of the mechanism of tryptophanyl transfer rna synthetase from yeast

Piel, N.; Freist, W.; Cramer, F.

Bioorganic Chemistry 12(1): 18-33


ISSN/ISBN: 0045-2068
Accession: 006569236

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Six analogs of tryptophanyl-adenylate, which is an important intermediate in the enzymatic synthesis of Trp-tRNATrp, were prepared. Four compounds, tryptophanyl-8-bromoadenylate, tryptophanyl-2-chloroadenylate, tryptophanyl-7-deazaadenylate and tryptophanyl-(N6-methyl)adenylate, contain modifications in the nucleobase moiety, while tryptophanyl-2'-deoxyadenylate and tryptophanyl-3'-deoxyadenylate were modified in the carbohydrate part of the molecule. Three of these analogs (2-chloro, 7-deaza, 2'-deoxy analogs) as well as ATP analogs with the same modifications were substrates in the aminoacylation reaction; 3 analogs (8-bromo, N6-methyl, 3'-deoxy analogs) were inactive as well as the corresponding ATP analogs. In the ATP/PPi pyrophosphate exchange in the absence of tRNA all ATP analogs except 8-bromo-ATP were substrates. The presence of tRNA reduced the number of ATP analogs being substrates to that number of substrates observed in the aminoacylation. The presence of tRNA is apparently responsible for an increase of specificity. The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP.alpha.S), adenosine 5'-O-(2-thiotriphosphate) (ATP.beta.S) and adenosine 5'-O-(3-thiotriphosphate) (ATP.gamma.S) were tested with various divalent metals as substrates in the pyrophosphate exchange reaction. The Sp diastereomer ATP.alpha.S is a substrate with Mg2+, whereas the Rp diastereomer is inactive. Both diastereomers are inactive in the presence of Zn2+. Since Zn2+ binds preferentially to the S atom, an explanation of these results is that the Mg2+ ion is not bound to the .alpha.-phosphate. Only the Sp isomer of the diastereomers of ATP.beta.S acts as substrate in the presence of Mg2+. The stereospecificity becomes reversed in the presence of Zn2+. ATP.gamma.S acts as substrate with both Mg2+ and Zn2+. The .DELTA. isomer of the .beta.,.gamma.-bidentate ATP-Mg2+ complex is apparently the substrate for this enzyme. From these results a molecular model of the ATP-Mg2+ complex in the active site can be derived in which the nucleotide is attached to the enzyme by interactions in which the 3'-OH and 6-NH2 group, 1 O atom of the .alpha.-P atom, and the coordinated Mg cation are all involved.

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