Atomic structure of the Serratia marcescens endonuclease at 1.1 A resolution and the enzyme reaction mechanism.

The three-dimensional crystal structure of Serratia marcescens endonuclease has been refined at 1.1 A resolution to an R factor of 12.9% and an R(free) of 15.6% with the use of anisotropic temperature factors. The model contains 3694 non-H atoms, 715 water molecules, four sulfate ions and two Mg(2+)-binding sites at the active sites of the homodimeric protein. It is shown that the magnesium ion linked to the active-site Asn119 of each monomer is surrounded by five water molecules and shows an octahedral coordination geometry. The temperature factors for the bound Mg(2+) ions in the A and B subunits are 7.08 and 4.60 A(2), respectively, and the average temperature factors for the surrounding water molecules are 12.13 and 10.3 A(2), respectively. In comparison with earlier structures, alternative side-chain conformations are defined for 51 residues of the dimer, including the essential active-site residue Arg57. A plausible mechanism of enzyme function is proposed based on the high-resolution S. marcescens nuclease structure, the functional characteristics of the natural and mutational forms of the enzyme and consideration of its structural analogy with homing endo-nuclease I-PpoI.

Three-dimensional structure of Serratia marcescens nuclease at 1.7 A resolution and mechanism of its action.

The three-dimensional crystal structure of Serratia marcescens (Sm) nuclease has been refined at 1.7 A resolution to the R-factor of 17.3% and R-free of 22.2%. The final model consists of 3678 non-hydrogen atoms and 443 water molecules. The analysis of the secondary and the tertiary structures of the Sm nuclease suggests a topology which reveals essential inner symmetry in all the three layers forming the monomer. We propose the plausible mechanism of its action based on a concerted participation of the catalytically important amino acid residues of the enzyme active site.

Structural characterization of extracellular ribonuclease of Bacillus polymyxa: amino acid sequence determination and spatial structure prediction.

The primary structure of extracellular Bacillus polymyxa ribonuclease (RNase Bpo) was established by mass spectroscopy analysis and automatic Edman degradation of the individual peptides obtained from protein digestion with Glu-specific protease V8. RNase Bpo consists of 111 amino acid residues, with a relative molecular weight of 12 607. RNase Bpo is a close structural homolog of RNases of B. amyloliquefaciens (RNase Ba) and B. intermedius (RNase Bi), the similarity of their primary structures being 68%. Molecular modelling of the structure of the complex of RNase Bpo with substrate analog d(CGAC) was performed and a spatial model based on the known crystal structure of RNase Ba complex with the corresponding nucleotide was constructed using the methods of interactive computer graphics and energy minimization. The differences in the primary and tertiary structures of the enzymes were analyzed in order to understand the substrate specificity of Bacillus RNases.

A novel guanyl-preferable ribonuclease of Bacillus polymyxa: isolation and characterization of the enzyme.

Three forms of the extracellular alkaline ribonuclease of B. polymyxa (RNases Bpo I, Bpo II and Bpo III) were obtained in the homogeneous states. Relative molecular weights, N- and C-terminal amino acid sequences of the proteins were determined. The two higher-molecular weight forms of the enzyme (RNases Bpo I and Bpo III) were shown to be precursor molecules which differed from the mature B. polymyxa RNase (RNase Bpo II) by the presence of a extra twelve and six amino acid residues at the amino terminus, respectively. The study of catalytic properties of the enzyme elucidated that the extent of specificity of B. polymyxa RNase in respect to highpolymeric substrates is different from that of other natural Bacillus RNases. Amino acid composition of B. polymyxa RNase was established and structural similarity within family of Bacillus RNases is discussed. B. polymyxa RNase was shown to be inhibited by intracellular protein inhibitor of B. amyloliquefaciens RNase with the dissociation constant of 2.7 x 10(-12) M.

Primary structure and catalytic properties of extracellular ribonuclease of Bacillus circulans.

A complete amino acid sequence of extracellular Bacillus circulans RNase was established and compared with a structure of B. amyloliquefaciens RNase. Gln15, Gly65 and Gln104 in B. amyloliquefaciens RNase were found to be replaced by Leu, Ala and Lys, respectively, in B. circulans RNase. Catalytic properties of B. circulans RNase were studied.

Two isoforms of Serratia marcescens nuclease. Crystallization and preliminary X-ray investigation of the enzyme.

Two isoforms of an extracellular endonuclease, nucleases Sm1 and Sm2, were purified from culture fluid of Serratia marcescens strain BIO MI by ligand-exchange chromatography on phosphocellulose and DEAE-Toyopearl 650S. The pI-values 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 nuclease Sm1 has been crystallized from ammonium sulfate solution by the vapour diffusion technique. The crystals belong to the space group P2(1)2(1)2(1) with unit cell constants a = 69.0, b = 106.7, c = 74.8 A, contain two molecules in an asymmetric unit, packing density Vm = 2.3 A/Da, and diffract to at least 1.5 A resolution. The Pt- and UO2-derivatives of the protein were obtained. Preliminary X-ray investigation of nuclease Sm2 crystals was carried out.

Amino acid sequence determination of guanyl-specific ribonuclease Sa from Streptomyces aureofaciens.

Using automated Edman degradation of two nonfractionated peptide mixtures of tryptic and staphylococcal protease digests of the protein, the complete amino acid sequence of the guanyl-specific ribonuclease Sa from Streptomyces aureofaciens was established. Ribonuclease Sa contains 96 amino acid residues (Mr 10,566). A 50% sequence homology of ribonuclease Sa to the guanyl-specific ribonuclease St from S. erythreus was found.

Express analysis of protein amino acid sequences. Primary structure of Penicillium chrysogenum 152A guanyl-specific ribonuclease.

The complete amino acid sequence of Penicillium chrysogenum 152A guanyl-specific RNase has been established using automated Edman degradation of two non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the protein. The RNase contains 102 amino acid residues: His2, Arg3, Asp7, Asn8, Thr5, Ser11, Glu4, Gln2, Pro4, Gly11, Ala13, Cys4, Val8, Ile3, Leu3, Tyr9, Phe5 (Mr 10 747).

Amino acid sequence and S-S bonds of Penicillium brevicompactum guanyl-specific ribonuclease.

The primary structure of Penicillium brevicompactum guanyl-specific RNase was determined. The enzyme consists of 102 amino acid residues, Mr 10801. The 4 cysteine residues of the RNase are linked in pairs by disulfide bonds: Cys2-Cys10, Cys6-Cys101. P. brevicompactum RNase structure is similar to RNase T1; the degree of homology is 66%.

The autophosphorylation reaction in the mechanism of activation of pig brain cyclic AMP-dependent protein kinase.

Autophosphorylation of cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was shown to occur via an intramolecular mechanism: the regulatory subunit undergoes phosphorylation only within the holoenzyme. The phospho form of the catalytic subunit has the capacity to phosphorylate the regulatory subunit. The phosphotransferase reaction and the reaction of autophosphorylation were found to proceed with the involvement of the same active site. The activation constant of phospho- and dephosphoprotein kinase under the influence of cyclic AMP and the dissociation constant of the cyclic AMP complex with phospho- and dephospho forms of the holoenzyme were estimated. Autophosphorylation was demonstrated to lead to almost complete dissociation of the holoenzyme under the influence of cyclic AMP. Circular dichroism spectra of the phosphorylated and non-phosphorylated forms of protein kinase were studied. The relative content of the secondary structure elements in proteins was estimated and conformational changes were detected in the enzyme upon its interaction with cycli AMP. The anti-conformation of the cyclic nucleotide fixed in the complex with the phospho form of the regulatory subunit is suggested.

Spectral properties of phosphopyridoxyl-Lys-7(-41)-ribonuclease A.

We have studied the spectral properties of RNAase A containing a phosphopyridoxyl residue at the epsilon-NH2 group of Lys-7 or Lys-14. The overall conformations of the native and modified enzymes were shown to be rather similar. All three proteins have similar circular dichroism spectra within the 220-300-nm region, and similar thermal transition temperatures. All the changes in the RNAase A molecule modified are located in close proximity to the alkylated lysine residue. The phosphopyridoxyl group of (P-Pxy)-epsilon-Lys-41-RNAase A is situated directly at the enzyme active site and is 25% butied in the protein globule. The P-pyridoxyl group of (P-Pxy)-epsilon-Lys-7-RNAase A was shown to be located in the vicinity of the active site and to be more exposed to the solvent. In the pyridoxyl phosphate absorption band, optical activity is induced in both proteins. Study of the pH dependence of the changes occurring in the circular dichroism and absorption spectra has shown that in the modified proteins, the pyridoxyl phosphate chromophore is rather sensitive to the ionic state of the surrounding medium and serves as a "reporter" group when the relationship between structure and function of the RNAase A active site is being investigated.