Norbert Piel

Synthesis of modified tryptophanyl-adenylates and of modified adenosine-triphosphates and their use as tools for elucidation of the mechanism of tryptophanyl-tRNA synthetase from yeast

Abstract Six analogs of tryptophanyl-adenylate, which is an important intermediate in the enzymatic synthesis of Trp-tRNA Trp , have been prepared. Four compounds, tryptophanyl-8-bromoadenylate, tryptophanyl-2-chloroadenylate, tryptophanyl-7-deazaadenylate and tryptophanyl-( N 6 -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; three analogs (8-bromo, N 6 -methyl, 3′-deoxy analogs) were inactive as well as the corresponding ATP analogs. In contrast, in the ATP PP i pyrophosphate exchange in the absence of tRNA all ATP analogs except 8-bromo-ATP were substrates. However, the presence of tRNA reduced the number of ATP analogs being substrates to that number of substrates observed in the aminoacylation. Therefore, it can be concluded that the presence of tRNA is responsible for an increase of specificity. The diastereomers of adenosine 5′- O -(3-thiotriphosphate) (ATPαS), adenosine 5′- O -(2-thiotriphosphate) (ATPβS), and adenosine 5′- O -(3-thiotriphosphate) (ATPγS) were tested with various divalent metals as substrates in the pyrophosphate exchange reaction. The S p diastereomer of ATPαS is a substrate with Mg 2+ , whereas the R p diastereomer is inactive. Both diastereomers are inactive in the presence of Zn 2+ . Since Zn 2+ binds preferentially to the sulfur atom, an explanation of these results is that the Mg 2+ ion is not bound to the α-phosphate. Only the S p isomer of the diastereomers of ATPβS acts as substrate in the presence of Mg 2+ . The stereospecificity becomes reversed in the presence of Zn 2+ . ATPγS acts as substrate with both Mg 2+ and Zn 2+ . These results suggest that the Δ isomer of the β,γ-bidentate ATP-Mg 2+ complex is the substrate for this enzyme. From these results a molecular model of the ATP-Mg 2+ 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-NH 2 group, one oxygen atom of the α-phosphorus atom, and the coordinated magnesium cation are all involved.

Phenylalanyl-tRNA synthetases from yeast cytoplasm and mitochondria. The presence of a carbohydrate moiety in the mitochondrial enzyme and immunological evidence for structural relationship.

Homogeneous yeast cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases (L-phenylalanine:tRNAPhe ligase (AMP-forming), EC 6.1.1.20) are analysed for structural differences. Only the large subunit of the mitochondrial enzyme is a glycoprotein with nearly 3% carbohydrate by weight. The carbohydrates present are: glucose, N-acetylglucosamine, mannose, galactose and N-acetylneuraminic acid. Removal of the sugar moieties yields an activity increase, but no significant change of sensitivity to proteolytic degradation. Antibodies to both homogeneous enzymes demonstrate a structural similarity for both types of subunit using the highly sensitive immunoblotting technique.

Synthesis of the oligodeoxyribonucleotide, d(CpTpGpGpApTpCpCpApG), and its substrate activity with the restriction endonuclease, BamHI

Abstract The synthesis of the DNA fragment, d(CpTpGpGpApTpCpCpApG), using a long-chain alkylamine-controlled pore glass bead polymer support, crystalline (9-phenyl-9-xanthenyl) nucleosides, and the bifunctional phosphorylating reagent, bis(1-benzotriazolyl)-2-chlorophenyl phosphate is described. For high coupling yields a mixed catalyst (1-methyl-imidazole + diisopropylethylamine) is required. The oligodeoxyribonucleotide shows activity with the DNA restriction endonuclease, Bam HI. Kinetic parameters have been determined for the reaction.