Transamination catalysed by tyrosine phenol-lyase from Citrobacter intermedius.

The interactions of tyrosine phenol-lyase with its substrates: L-tyrosine and L-serine, and the competitive inhibitors: L-alanine, L-phenylalanine, L-m-tyrosine, were studied. It was demonstrated that the enzyme catalyzed a half-transamination reaction between substrates or inhibitors and the protein-bound pyridoxal phosphate. The products of this side-reaction, pyridoxamine phosphate and the respective keto acids, were identified. The kinetic parameters were determined for beta-elimination of L-tyrosine and of L-serine, and for the transamination of L-serine and the inhibitors used. The transfer of the amino group to the coenzyme takes place in the direction from amino acid to pyridoxal phosphate, but not in the opposite direction, i.e. the transamination is irreversible.

Spin-labelled analogues of GDP and GTP as site-specific reporter groups for guanosine nucleotide-binding proteins.

New derivatives of GDP and GTP have been synthesized for the spectroscopic investigation of the interaction between guanosine nucleotides and guanosine nucleotide-binding proteins. The 3'-hydroxyl group in these nucleotides was replaced by a 3'-amino group, which was further derivatized by the introduction of a spin-label reporter group. The biological activity of 3'SL-GDP and 3'SL-GTP could be demonstrated by measuring the interaction of these spin-labelled derivatives with bacterial elongation factor Tu. The amino modification and spin labelling only slightly influenced the affinity of the guanosine nucleotides for EF-Tu from Escherichia coli or Thermus thermophilus. Electron paramagnetic resonance (EPR) measurements revealed a strong immobilization of the labelled nucleotides upon binding to T. thermophilus EF-Tu. Significant differences between the spectra of EF-Tu X 3'SL-GDP, EF-Tu X 3'SL-GTP and aminoacyl-tRNA X EF-Tu X 3'SL-GTP ternary complexes were observed. Our data demonstrate that spin-labelled guanosine nucleotides can be used as sensitive spectroscopic probes for the investigation of the local environment of the nucleotide-binding site during distinct functional states of a guanosine nucleotide-binding protein.

2',3'-Dideoxy-3' aminonucleoside 5'-triphosphates are the terminators of DNA synthesis catalyzed by DNA polymerases.

It is shown that 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates with adenine, guanine, cytosine and thymine bases are effective inhibitors of DNA polymerase I, calf thymus DNA polymerase alpha and rat liver DNA polymerase beta. The effect of the above-mentioned compounds is markedly higher than corresponding action of the well-known DNA synthesis inhibitors arabinonucleoside 5'-triphosphates and 2',3'-dideoxynucleoside 5'-triphosphates. 2',3'-dideoxy-3'-aminonucleoside 5'-monophosphate residues incorporate into the 3'-terminus of the primer and terminate the DNA chain elongation. The possibility of using 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates as terminators for DNA sequencing by the polymerization method is demonstrated.

Aminonucleosides and their derivatives. IV. Synthesis of the 3'-amino-3'-deoxynucleoside 5'-phosphates.

A new procedure has been developed for the synthesis of 3'-amino-3'-deoxyribonucleosides of adenine, cytosine and uracil by condensing the trimethylsilylated bases with peracylated 3-azido-3-deoxyribose derivative. The azido group could subsequently be reduced to amino. The 5'-phosphates of these nucleosides have been prepared and the analogues have been tested for their ability to stimulate the ribosome-catalyzed reaction of 3'(2')-O-(N-formylmethionyl) adenosine 5'-phosphate with phenylalanyl-tRNA.

Synthesis of acylamino acid esters of nucleoside 5'-phosphates and their investigation with PMR and CD spectra.

The acylamino acid esters of nucleoside 5'-phosphates are synthesized via condensation of N-(N'-acylaminoacyl) imidazoles with nucleoside 5'-phosphates. The PMR and CD spectra of the esters obtained are studied. The 3'-isomers of the substances under study are observed to have a shift in the conformational N in equilibrium S equilibrium of the carbohydrate moiety in favour of the S-form as compared to the initial nucleosides and their 2'-acyl esters.

Thioacyl esters of nucleotides: synthesis of 3'(2')-O-thiobenzoyl nucleoside 5'-phosphates.

The synthesis of 3'(2')-O-thiobenzoyl nucleoside 5'-phosphates based on the condensation of N-(thiobenzoyl)-imidazole with nucleside 5'-phosphates was carried out. The UV absorption spectra, CD spectra, PMR spectra and chromatographic and electrophoretic characteristics of synthesized compounds were obtained. By means of PMR it was shown that the 2':3' isomer ratio in water at ambient temperature is about 2:3.