Mobile Loop in the Active Site of Metallocarboxypeptidases as an Underestimated Determinant of Substrate Specificity.

It is generally accepted that the primary specificity of metallocarboxypeptidases is mainly determined by the structure of the so-called primary specificity pocket. However, the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT) with the primary specificity pocket fully reproducing the one in pancreatic carboxypeptidase B (CPB) retained the broad, mainly hydrophobic substrate specificity of the wild-type enzyme. In order to elucidate factors affecting substrate specificity of metallocarboxypeptidases and the reasons for the discrepancy with the established views, we have solved the structure of the complex of the CPT G215S/A251G/T257A/D260G/T262D mutant with the transition state analogue N-sulfamoyl-L-phenylalanine at a resolution of 1.35 Å and compared it with the structure of similar complex formed by CPB. The comparative study revealed a previously underestimated structural determinant of the substrate specificity of metallocarboxypeptidases and showed that even if substitution of five amino acid residues in the primary specificity pocket results in its almost complete structural correspondence to the analogous pocket in CPB, this does not lead to fundamental changes in the substrate specificity of the mutant enzyme due to the differences in the structure of the mobile loop located at the active site entrance that affects the substrate-induced conformational rearrangements of the active site.

Three-dimensional structure of carboxypeptidase T from Thermoactinomyces vulgaris in complex with N-BOC-L-leucine.

The 3D structure of recombinant bacterial carboxypeptidase T (CPT) in complex with N-BOC-L-leucine was determined at 1.38 Å resolution. Crystals for the X-ray study were grown in microgravity using the counter-diffusion technique. N-BOC-L-leucine and SO4(2-) ion bound in the enzyme active site were localized in the electron density map. Location of the leucine side chain in CPT-N-BOC-L-leucine complex allowed identification of the S1 subsite of the enzyme, and its structure was determined. Superposition of the structures of CPT-N-BOC-L-leucine complex and complexes of pancreatic carboxypeptidases A and B with substrate and inhibitors was carried out, and similarity of the S1 subsites in these three carboxypeptidases was revealed. It was found that SO4(2-) ion occupies the same position in the S1' subsite as the C-terminal carboxy group of the substrate.

[A novel endogeneous inhibitor from hepatopancreas of Kamchatka crab (Paralithodes camtschaticus)].

A novel endogeneous inhibitor from hepatopancreas of Kamchatka crab (Paralithosed camtschaticus) was isolatyed. The inhibitor was purifeid through fractional affinity chromatography on gramicidin-diasorb followed by gel-filtration at Sephadex G-100. The inhibitor PC is a protein (M, 66 kDa) and active against serine collagenolytic protease PC at temperature optimum 15-20 degrees C, stable at 4-40 degrees C and was completely inactivated after heating to 50 degrees C and higher. 0.9-20% NaCl is necessary for its inhibitor activity. The inhibitor was found to slow down cell spreading in vitro in cell type-dependent manner. Fibroblasts are most prone to inhibitory effect, epithelial tumor derived cells show medium susceptibility, while fibrosarcoma cells were not affected.

X-ray investigation of gene-engineered human insulin crystallized from a solution containing polysialic acid.

Attempts to crystallize the noncovalent complex of recombinant human insulin with polysialic acid were carried out under normal and microgravity conditions. Both crystal types belonged to the same space group, I2(1)3, with unit-cell parameters a = b = c = 77.365 A, alpha = beta = gamma = 90.00 degrees. The reported space group and unit-cell parameters are almost identical to those of cubic insulin reported in the PDB. The results of X-ray studies confirmed that the crystals obtained were cubic insulin crystals and that they contained no polysialic acid or its fragments. Electron-density maps were calculated using X-ray diffraction sets from earth-grown and microgravity-grown crystals and the three-dimensional structure of the insulin molecule was determined and refined. The conformation and secondary-structural elements of the insulin molecule in different crystal forms were compared.

Three-dimensional structures of L-asparaginase from Erwinia carotovora complexed with aspartate and glutamate.

The crystal structures of Erwinia carotovora L-asparaginase complexed with L-aspartate and L-glutamate were determined at 1.9 and 2.2 A, respectively, using the molecular-replacement method and were refined to R factors of about 21% in both cases. The positions of the ligands in the active site were located. A comparison of the new structures with the known structures of Escherichia coli L-asparaginase and Er. chrysanthemi L-asparaginase was performed. It was found that the arrangement of the ligands practically coincides in all three enzymes. The peculiarities of the quaternary structure of the enzyme, the possible role of water molecules in the enzyme action and the conformational changes during the catalyzed reaction are discussed.

Modification of substrate-binding site of glutamyl endopeptidase from Bacillus intermedius.

Glutamyl endopeptidases (GEPs) are serine proteases belonging to the chymotrypsin structural family. Although the family as a whole has been described in detail, the molecular mechanism underlying strict substrate specificity of GEPs remains unclear. The most popular hypothesis attributes the key role in recognition of the charged substrates by GEPs to the conserved amino acid His213 (chymotrypsin numbering system). In order to test the role of this residue in the substrate specificity, we obtained a GEP from Bacillus intermedius with an amino acid substitution (His213-Thr) and studied its catalytic properties. Such modification proved not to affect the primary specificity of the enzyme. The introduced substitution had little effect on the Michaelis constant (Km increased 4.9 times) but considerably affected the catalytic constant (kcat decreased 615 times). The obtained data suggest that the conserved His213 residue in Bacillus GEPs is not a key element determining their primary substrate specificity.

Glutamyl endopeptidase of Bacillus intermedius strain 3-19. Purification, properties, and crystallization.

A homogeneous glutamyl endopeptidase splitting peptide bonds of glutamic and, rarely, of aspartic acid residues in peptides and proteins was isolated from Bacillus intermedius 3-19 culture filtrate using chromatography on CM-cellulose and Mono S. The enzyme molecular mass is 29 kD and the pI is 8.4. The proteinase is inhibited by DFP. The enzyme, like other glutamyl endopeptidases, reveals two pH optima (pH 7.5 and 9.0) for casein and one (pH 8.0) for Z-Glu-pNA hydrolysis. The K(m) for the hydrolysis of the latter substrate is 6 mM. The enzyme activity is optimal at 55 degrees C. The enzyme is stable in the pH range 6.5-11.0. Its N-terminal sequence shows 56% coinciding residues when compared with that of Bacillus licheniformis glutamyl endopeptidase. Crystal prisms or plates 0.25-0.3 x 0.15 x 0.07-0.1 mm have been grown using the vapor diffusion technique in a hanging drop followed by macroseeding. The crystals belong to the space group B2 with the following unit cell parameters: a = 69.59 A; b = 61.61 A; c = 56.11 A; gamma = 117.57 degrees. The X-ray data set to 1.7 A resolution has been collected on an automatic synchrotron (EMBL Hamburg Station).

The binding of carbon monoxide and nitric oxide to leghaemoglobin in comparison with other haemoglobins.

Haemoglobins have the ability to discriminate between oxygen and other diatomic molecules. To further understanding of this process the X-ray crystal structures of carbonmonoxy and nitrosyl-leghaemoglobin have been determined at 1.8 A resolution. The ligand geometry is discussed in detail and the controversial issue of bent versus linear carbon monoxide binding is addressed. The bond angle of 160 degrees for CO-leghaemoglobin is in conflict with recent spectroscopy results on myoglobin but is consistent with angles obtained for myoglobin X-ray crystal structures. In contrast to the numerous carbon monoxide studies, very little stereochemical information is available for the nitric oxide adduct of haemoglobin. This is provided by the X-ray structure of NO-leghaemoglobin, which conforms to expected geometry with an Fe-NO angle of 147 degrees and a lengthened iron-proximal histidine bond. Thus crystallographic evidence is given for the predicted weakening of this bond on the binding of nitric oxide.

X-ray structure of yeast inorganic pyrophosphatase complexed with manganese and phosphate.

The three-dimensional structure of the manganese-phosphate complex of inorganic pyrophosphatase from Saccharomyces cerevisiae has been refined to an R factor of 19.0% at 2.4-A resolution. X-ray data were collected from a single crystal using an imaging plate scanner and synchrotron radiation. There is one dimeric molecule in the asymmetric unit. The upper estimate of the root-mean-square coordinate error is 0.4 A using either the delta A plot or the superposition of the two crystallographically independent subunits. The good agreement between the coordinates of the two subunits, which were not subjected to non-crystallographic symmetry restraints, provides independent validation of the structure analysis. The active site in each subunit contains four manganese ions and two phosphates. The manganese ions are coordinated by the side chains of aspartate and glutamate residues. The phosphate groups, which were identified on the basis of their local stereochemistry, interact either directly or via water molecules with manganese ions and lysine, arginine, and tyrosine side chains. The phosphates are bridged by two of the manganese ions. The outer phosphate is exposed to solvent. The inner phosphate is surrounded by all four manganese ions. The ion-binding sites are related to the order of binding previously established from kinetic studies. A hypothesis for the transition state of the catalytic reaction is put forward.

The structure of deoxy- and oxy-leghaemoglobin from lupin.

The leghaemoglobins have oxygen affinities 11 to 24 times higher than that of sperm whale myoglobin, due mainly to higher rates of association. To find out why, we have determined the structures of deoxy- and oxy-leghaemoglobin II of the lupin at 1.7 A resolution. Results confirm the general features found in previous X-ray analyses of this protein. The unique feature that has now emerged is the rotational freedom of the proximal histidine. In deoxy-leghaemoglobin the imidazole oscillates between two alternative orientations, eclipsing either the lines N1-N3 or N2-N4 of the porphyrin; in oxy-leghaemoglobin it is fixed in a staggered orientation. The iron atom moves from a position 0.30 A from the plane of the pyrrole nitrogen atoms in deoxy- to a position in the plane in oxy-leghaemoglobin while the Fe- bond distance remains constant at 2.02 A. The Fe-O-O angle is 152 degrees, as in human haemoglobin. The oxygen is hydrogen-bonded to the distal histidine at N epsilon 2-O1 and N epsilon 2-O2 distance of 2.95 A and 2.68 A, respectively. The porphyrin is ruffled equally in deoxy- and oxy-leghaemoglobins, due to rotations of the pyrrols about the N-Fe-N bonds, causing the methine bridges to deviate by up to 0.32 A from the mean porphyrin plane. The only feature capable of accounting for the high on-rate of the reaction with oxygen are the mobilities of the proximal histidine and distal histidine residues in deoxy-leghaemoglobin. The eclipsed positions of the proximal histidine in deoxy-leghaemoglobin maximize steric hindrance with the porphyrin nitrogen atoms and minimize pi-->p electron donation, while its staggered position in oxy-leghaemoglobin reverses both these effects. Together with the oscillation of the imidazole between the two orientations, these two factors may reduce the activation energy for the reaction of leghaemoglobin with oxygen. The distal histidine is in a fixed position in the haem pocket in the crystal, but must be swinging in and out of the pocket at a high rate in solution to allow the oxygen to enter.

Crystal structure of thermitase at 1.4 A resolution.

The crystal structure of thermitase, a subtilisin-type serine proteinase from Thermoactinomyces vulgaris, was determined by X-ray diffraction at 1.4 A resolution. The structure was solved by a combination of molecular and isomorphous replacement. The starting model was that of subtilisin BPN' from the Protein Data Bank, determined at 2.5 A resolution. The high-resolution refinement was based on data collected using synchrotron radiation with a Fuji image plate as detector. The model of thermitase refined to a conventional R factor of 14.9% and contains 1997 protein atoms, 182 water molecules and two Ca ions. The tertiary structure of thermitase is similar to that of the other subtilisins although there are some significant differences in detail. Comparison with subtilisin BPN' revealed two major structural differences. The N-terminal region in thermitase, which is absent in subtilisin BPN', forms a number of contacts with the tight Ca2+ binding site and indeed provides the very tight binding of the Ca ion. In thermitase the loop of residues 60 to 65 forms an additional (10) beta-strand of the central beta-sheet and the second Ca2+ binding site that has no equivalent in the subtilisin BPN' structure. The observed differences in the Ca2+ binding and the increased number of ionic and aromatic interactions in thermitase are likely sources of the enhanced stability of thermitase.

[Crystal structure of thermitase and stability of subtilisins]. / Kristallicheskaia struktura termitazy i stabil'nost' subtilizinov.

Crystal structure of thermitase, a serine proteinase from Thermoactinomyces vulgaris, has been determined by X-ray diffraction at 1.4 A resolution. The atomic model of thermitase refined to an R-factor of 0.149 contains 1997 protein atoms, 182 water molecules and 2 Ca2+ ions. The tertiary structure of thermitase is similar to that of subtilisin BPN'. The greatest variations are connected with insertions and deletions in the amino acid sequence, which are located on the surface of the molecule. Higher thermostability of thermitase can be explained in terms of the three-dimensional structure. The Ca2+ ions, bound to the protein molecule, as well as the ionic and hydrophobic interactions are supposed to give the main contribution to the stabilization of the structure.

Crystal structure of thermitase from Thermoactinomyces vulgaris at 2.2 A resolution.

The crystal structure of thermitase from Thermoactinomyces vulgaris has been determined by x-ray diffraction at 2.2 A resolution. The structure was solved by a combination of single isomorphous replacement and molecular replacement methods. The structure was refined to a conventional R factor of 0.24 using restrained least square procedures CORELS and PROLSQ. The tertiary structure of thermitase is similar to that of subtilsin BPN'. The greatest differences between these structures are related to the insertions and deletions in the sequence.

Kinetic characterization of a thermostable inorganic pyrophosphatase from Thermus thermophilus.

A kinetic characterization was performed for inorganic pyrophosphatase from Thermus thermophilus. The optimum activity with Mg2+ as the activating metal ion lies in a pH range between 8.3 and 9.5. The hydrolysis of inorganic pyrophosphate is also activated by Zn2+, Mn2+, and Co2+. Calcium ions are not activating at all. Tripolyphosphate is another substrate hydrolyzed by the enzyme but only with Zn2+ as the activating metal ion. Other potential substrates like ATP and cyclic metaphosphates are not hydrolyzed even at high enzyme concentrations. Computer modelling of kinetic data obtained from activity measurements with different total magnesium ion and pyrophosphate concentrations confirms a kinetic model which was shown to be valid also for inorganic pyrophosphatases from other microbial sources. The corresponding parameter values are given. The inorganic pyrophosphatase from T. thermophilus exhibits extremely high thermostability which is decreased by addition of EDTA indicating a stabilizing effect of divalent metal ions.

[Physico-chemical characteristics of catalases from Micrococcus sp. n]. / Fiziko-khimicheskie kharakteristiki katalaz iz Micrococcus sp. n.

The physico-chemical properties of heme-containing and non-heme catalases isolated from the cell culture of Micrococcus sp. n. grown under intensive aeration were studied. The enzyme preparations were homogenous during polyacrylamide disc electrophoresis. The spectral and functional properties of the enzymes (e. g. specific activity, subunit molecular weight, quaternary structure, amino acid composition, immunoprecipitability, N-terminal amino acid sequences) were investigated. Monocrystals of non-heme catalase applicable for an X-ray analysis were grown and examined by X-ray spectroscopy. Both enzymes were stable upon storage in 40% ammonium sulfate for 2 months and resistant to lyophylization without any significant loss of their activity. Non-heme catalase is apparently an independent enzyme which is not derived from heme-containing catalase via dissociation, limited proteolysis or heme cleavage.

[Interaction of terbium ions with inorganic pyrophosphatase from bakers' yeast: characterization of the binding sites]. / Wechselwirkung von Tb3+ mit anorganischer Pyrophosphatase aus Bäckerhefe: Charakterisierung der Bindungsorte.

Terbium ions bind with a 2:1 stoichiometry per subunit to inorganic pyrophosphatase from bakers' yeast (EC 3.6.1.1) as measured by an increase of terbium fluorescence. The Tb3+ inhibition of the Mg2+ activated pyrophosphate hydrolysis is caused by a competitive binding at the substrate site of the active centre. The second Mg2+ binding site--the so-called "stabilization site"--is discussed as an additional binding site for Tb3+. Thereby, Tb3+ causes also a stabilization of the enzyme against heat denaturation. The dissociation constants of the terbium-pyrophosphatase interaction are in the micromolar range.

[Separation of leghemoglobin from the nodules of lupine, Lupinus luteus L., into components]. / Razdelenie na komponenty leggemoglobina iz kluben'kov liupina Lupinus luteus L

The crude leghaemoglobin suspension from root nodules of yellow lupine (Lupinus luteus L.) was separated into five fractions: I, IIa and IIb, IIIa and IIIb using the chromatography on DEAE-cellulose (numbered following the elution order). The visible absorption spectra show that fractions I, IIa and IIIa are met-leghaemoglobins while IIb and IIIb are oxy-forms. IIb and IIIb were converted to IIa and IIIa respectively when oxidized with potassium ferricyanide. The determination of two first N-terminal amino acids confirms the identity of pairs IIa-IIb and IIIa-IIIb. Three components of leghaemoglobin were obtained having the following N-terminal amino acids: Gly-Val- (the first), Gly-Ala- (the second), Ala-Val- (the third). The molecular weights obtained by Archibald's method are 17 900, 17 600, 18 800 respectively. The second leghaemoglobin species which contains more than 50% of the starting protein mixture was used to grow crystals for X-ray study of the tertiary structure of this protein.

Microcalorimetry of thermal denaturation of pepsin and trypsin in the crystal state.

Heating of pepsin and trypsin crystals was studied by scanning microcalorimetry. A sharp decrease in temperature, halfwidth and heat of transition with a decrease in heating rate was discovered. It was shown that thermal transition is connected only with the denaturation of protein molecules in the crystal and not accompanied by the crystal disintegration into separate molecules.

The haem-accessibility in leghaemoglobin of Lupinus luteus as observed by proton magnetic relaxation.

Using the solvent-protons' longitudinal magnetic relaxation rates (p.m.r.) for Lupinus luteus leghaemoglobin derivatives the accessibility of the haem has been evaluated by our "stereo-chemical p.m.r. titration" method with nonexchangeable protons of aliphatic lower alcohols in otherwise deuterated solutions. The haem in leghaemoglobin is more accessible and its protein environment more flexible compared with vertebrate haemoglobins. The correlation time in aquometleghaemglobin aqueous solution has been determined by measuring the frequency dispersion of the p.m.r. rates between 6.1 and 93 MHZ. Taking into account the measured value of tauc = (7.7 +/- 0.5 x 10(-10) s the iron-to-proton inter-spin distances have been calculated. The significance of these distances as well as the electronic g-factor anisotrophy for elucidation of fine structural details of the haem-environment are discussed.