[The conformational dynamic of the tetramer hemoglobin molecule as revealed by hydrogen exchange. II. Influence of the intersubunit contact splitting].

The rate of the H-D exchange of the peptide NH atoms of the isolated alpha and beta subunits of human Hb were studied at the pH range 5.5-9.0 and 20 degrees C by the IR spectroscopy. The factor retardation of the exchange rate of subunits -P in the range -10(2)-10(7). In comparison with tetramer Hb the probability of local fluctuations (1/P) is increased to a slightly greater extent for the monomeric alpha subunits then for the tetramer beta subunits. Unlike Hb oxygenation of subunits does not influence on the probability of the local fluctuations and subunits have no the pH-dependent change of the value 1/P observable for the ligand Hb. The possible mechanisms of the overall intensification of the local fluctuations upon the splitting of the Hb tetrameric contacts between subunits are discussed with the inviting of the structural crystallographic data.

[The conformational dynamic of the tetramer hemoglobin molecule as revealed by hydrogen exchange. I. Influence pH, temperature and ligand binding].

The rate of the H-D exchange of the peptide NH atoms of the different forms of human Hb was studied at the range of pH 5-10 and temperature 10-63 degrees C by the IR spectroscopy. The pH-dependence of the H-D exchange rate is accordance with the EX2 mechanism. Two pH-dependent conformers of ligand forms of Hb existes at 10-30 degrees C with lower probability of local fluctuations of the alkaline conformer. The difference between two conformers vanishes at 40 degrees C with the appearance of the third conformer with higher probability of local fluctuations. The deoxyHb at 20 degrees C and pH range 6-9 has no pH-dependent conformers and the probability of local fluctuations is considerably reduced in comparison to the acid conformer of ligand Hb. Upon the destabilization of the ligand Hb structure by the pH decreasing to 5.0 at 20 degrees C or the temperature increasing up to 50-60 degrees C at pH 7.1 the global fluctuations of the native structure are intensified providing the H-D exchange of the slowest exchanging NH atoms. The nature of the local and global fluctuations and possible similarity between the two pH-dependent conformers of ligand Hb and its functional R and R2 states revealed by the X-ray analysis and NMR spectroscopy were discussed.

[A comparative structure-function analysis and molecular mechanism of action of endonucleases from Serratia marcescens and Physarum polycephalum]. / Sravnitel'nyi strukturno-funktsional'nyi analiz éndonukleaz Serratia marcescens i Physarum polycephalum i molekuliarnyi mekhanizm ikh deistviia.

Structural and functional characteristics were compared for wild-type nuclease from Serratia marcescens, which belongs to the family of DNA/RNA nonspecific endonucleases, its mutational forms, and the nuclease I-PpoI from Physarum polycephalum, which is a representative of the Cys-His box-containing subgroup of the superfamily of extremely specific intron-encoded homing DNases. Despite the lack of sequence homology and the overall different topology of the Serratia marcescens and I-PpoI nucleases, their active sites have a remarkable structural similarity. Both of them have a unique magnesium atom in the active site, which is a part of the coordinatively bonded water-magnesium complex involved in their catalytic acts. In the enzyme-substrate complexes, the Mg2+ ion is chelated by an Asp residue, coordinates two oxygen atoms of DNA, and stabilizes the transition state of the phosphate anion and 3'-OH group of the leaving nucleotide. A new mechanism of the phosphodiester bond cleavage, which is common for the Serratia marcescens and I-PpoI nucleases and differs from the known functioning mechanism of the restriction and homing endonucleases, was proposed. It presumes a His residue as a general base for the activation of a non-cluster water molecule at the nucleophilic in line displacement of the 3'-leaving group. A strained metalloenzyme-substrate complex is formed during hydrolysis and relaxes to the initial state after the reaction. The English version of the paper.

[X-ray structural analysis of magnesium-containing Serratia marcescens endonuclease]. / Rentgenostrukturnyi analiz magniisoderzhashchei éndonukleazy Serratia marcescens.

The three-dimensional crystal structure of the DNA/RNA nonspecific endonuclease from Serratia marcescens was refined at the resolution of 1.07 A to R factor of 12.4% and Rfree factor of 15.3% using the anisotropic approximation. The structure includes 3924 non-hydrogen atoms, 715 protein-bound water molecules, and a Mg2+ ion in each binding site of each subunit of the nuclease homodimeric globular molecule. The 3D topological model of the enzyme was revealed, the inner symmetry of the monomers in its N- and C-termini was found, and the local environment of the magnesium cofactor in the nuclease active site was defined. Mg2+ ion was found to be bound to the Asn119 residue and surrounded by five associated water molecules that form an octahedral configuration. The coordination distances for the water molecules and the O delta 1 atom of Asn119 were shown to be within a range of 2.01-2.11 A. The thermal factors for the magnesium ion in subunits are 7.08 and 4.60 A2, and the average thermal factors for the surrounding water molecules are 11.14 and 10.30 A2, respectively. The region of the nuclease subunit interactions was localized, and the alternative side chain conformations were defined for 51 amino acid residues of the nuclease dimer.

[Primary structure and catalytic properties of extracellular ribonuclease from Bacillus circulans]. / Pervichnaia struktura i kataliticheskie svoistva vnekletochnoi ribonukleazy Bacillus circulans.

A comparative research of individual peptide structures obtained after hydrolysing of Bacillus circulans and B. amyloliquefaciens RNases by the Glu-specific staphylococcal protease was carried out by means of mass-spectrometry and Edman degradation methods. A complete amino acid sequence of B. circulans RNase was determined. Gln15, Gly65 and Gln104 residues in B. amyloliquefaciens RNase were found to be substituted by Leu, Ala and Lys residues in B. circulans RNase, respectively. Catalytic properties of the B. circulans RNase in transesterification reactions with poly- and oligonucleotides as substrates were investigated.

[Extracellular alkaline ribonucleases produced by Bacilli strains from the permafrost of the Kolyma lowland]. / Vnekletochnye shchelochnye ribonukleazy batsill vechnomerzlykh gruntov kolymskoi nizmennosti.

Aerobic spore-forming bacteria were isolated from the permafrost of the Kolyma lowland. Two strains of bacilli are shown to produce a relatively large amount of extracellular low-molecular weight alkaline RNases. The N-terminal amino acid sequences of the RNases secreted by these strains are similar. This suggests that the protein sequences of the RNases of Bacillus species have been conserved in the course of evolution.

[Complete primary structure of Bacillus thuringiensis extracellular ribonuclease]. / Polnaia pervichnaia struktura vnekletochnoi ribonukleazy bakterii Bacillus thuringiensis.

Complete primary structure of an extracellular low molecular mass ribonuclease of Bacillus thuringiensis was determined using Edman degradation and mass-spectrometry analysis of individual peptides obtained after hydrolysis of the protein by cyanogen bromide and staphylococcal protease. The peptides were isolated and purified by HPLC and denaturing PAGE. The enzyme consists of 109 amino acid residues (Asp 8, Asn 6, Thr 6, Ser 10, Glu 3, Gln 1, Pro 3, Gly 9, Ala 12, Val 7, Ile 7, Leu 7, Tyr 7, Phe 4, His 1, Arg 10, Trp 3 and Lys 5) and has a molecular weight of 12182 Da. A single difference was detected between primary structures of the enzyme and an extracellular ribonuclease of B. intermedius.

[Two forms of extracellular low molecular weight Bacillus sp. BCF 247 ribonuclease. Isolation and characteristics of the protein]. / Dve formy vnekletochnoi nizkomolekuliarnoi ribonukleazy Bacillus sp. BCF 247. Vydelenie i kharakteristika belka.

Two homogeneous samples of low molecular mass RNAase (RNAases Bci I and Bci II) were obtained from cultural filtrates of spore-forming bacteria strain Bacillus sp. BCF 247 isolated from permafrost soils. The yields of RNAases Bci I and Bci II were 17% and 16% at the 17388- and 15376-fold degree of purification, respectively. Both enzymes have a close specific activity which is equal to approximately 4.7 x 10(-5) activity units per mg of protein. The relative molecular masses of the isolated proteins were determined and their N-terminal amino acid sequences identified. It was shown that the higher molecular mass sample of the enzyme is a pro-RNAase which, in contrast with the mature protein, contains an additional decapeptide segment in the N-terminal part of its molecule. The structure of RNAase Bci was compared with that of RNAases obtained from other Bacillus species; its ability to interact with a natural intracellular inhibitor of B. amyloliquefaciens RNAase was demonstrated.

[Isolation and characteristics of intracellular nuclease isoforms from Serratia marcescens]. / Vydelenie i kharakteristika izoform vnekletochnoi nuklezy Serratia marcescens.

Two enzyme forms were isolated from the commercial preparation of extracellular endonuclease of Serratia marcescens strain B10 M1. The chromatographic and electrophoretic properties, isoelectric points and N-terminal amino acid residues are different for both enzymes. At the final step of the purification procedure including ion-exchange chromatography on phospho- and DEAE-cellulose columns the yields of nucleases Sm1 and Sm2 were 13% and 25%, respectively. No significant differences were found in the specific activities of nucleases Sm1 and Sm2 (3.6 x 10(6) and 4.0 x 10(6) un. act./mg of protein). A comparative analysis of tryptic nuclease hydrolysate peptides was carried out. The amino acid sequences of some polypeptide segments of the proteins were determined. The structural similarity of the enzyme was established and the amino terminal regions of the proteins were identified. The localization of the disulfide bonds in the molecules of the both nucleases was determined. The similarity of nucleases Sm1 and Sm2 strain B10 M1 to S. marcescens endonucleases obtained from other strains was demonstrated.

[Isolation of isoforms and crystallization of endonucleases of Serratia marcescens]. / Razdelenie izoform i kristallizatsiia éndonukleazy Serratia marcescens.

Two isoforms of an extracellular endonuclease, nuclease Sm1 and nuclease Sm2, were isolated from the culture filtrate of Serratia marcescens strain B10 M1 by the ligand-exchange chromatography on iminodiacetate-agarose in Cu2(+)-form, and chromatography on phosphocellulose and DEAE-Toyopearl 650S. The pI for nucleases Sm1 and Sm2 were found to be 7.1 and 6.7, respectively. The amino acid analysis and N-terminal amino acid sequencing of the proteins showed a significant degree of homology between the enzymes. The secondary structure of nuclease Sm2 was calculated. Crystals of nuclease Sm2 were obtained with the space group P2(1)2(1)2(1), a 69.0; b 106.7; c 74.8 A.

[Ribonuclease Fl1 from Fusarium lateriticum. Isolation, substrate specificity and amino acid sequence]. / Ribonukleaza Fl1 Fusarium lateriticum. Vydelenie, substratnaia spetsifichnost' i aminokislotnaia posledovatel'nost'.

Extracellular RNase Fl1 has been purified from the culture filtrate of Fusarium lateritium. The enzyme has been obtained in the electrophoretically homogeneous state with the yield about 90% and 300 fdd degree of purification. RNase Fl1 is a guanyl specific enzyme (EC 3.1.27.3) with the specific activity on RNA 1420 units/mg of protein. The total primary structure of the RNase has been determined by the automated Edman degradation of two non-fractionated peptide hydrolysates produced by trypsin and Staphylococcus aureus protease and of the hydroxylamine cleavage products of the protein. It was shown that hydroxylamine converts the RNase Fl1 N-terminal residue, pyroglutamic acid, into the hydroxyamic acid derivative sensitive to Edman degradation. RNase Fl1 consists of 105 amino acid residues (Mr 10,852) and is a structural homologue of the Fus. moniliforme RNase F1, differing from the latter by 15 amino acid substitutions outside the enzyme active site.