Biomedical subjects
Publications and source records attributed to D M Blow.
Crystals of an inhibitor of trypsin and tissue plasminogen activator from seeds of the legume Erythrina caffra have been obtained by vapour diffusion. The crystals belong to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22) with cell parameters 73.4 A and 143.0 A. There is one molecule in the asymmetric unit. The crystals diffract to beyond 2.5 A resolution.
The crystal structure of tyrosyl-tRNA synthetase (EC 6.1.1.1) from Bacillus stearothermophilus has been refined to a crystallographic R-factor of 22.6% at 2.3 A resolution using a restrained least-squares procedure. In the final model the root-mean-square deviation from ideality for bond distances is 0.018 A and for angle distances is 0.044 A. Each monomer consists of three domains: an alpha/beta domain (residues 1 to 220) containing a six-stranded beta-sheet, an alpha-helical domain (248 to 318) containing five helices, and a disordered C-terminal domain (319 to 418) for which the electron density is very weak and where it has not been possible to trace the polypeptide chain. Complexes of the enzyme with the catalytic intermediate tyrosyl adenylate and the inhibitor tyrosinyl adenylate have also been refined to R-factors of 23.9% at 2.8 A resolution and 21.0% at 2.7 A resolution, respectively. Formation of the complexes results in some crystal cracking, but there is no significant difference in the conformation of the polypeptide chain of the three structures described here. The relative orientation of the alpha/beta and alpha-helical domains is similar to that previously observed for the "A" subunit of a deletion mutant lacking the C-terminal domain. Differences between these structures are confined to surface loops that are involved in crystal packing. Tyrosyl adenylate and tyrosinyl adenylate bind in similar conformations within a deep cleft in the alpha/beta domain. The tyrosine moiety is in the equivalent position to that occupied by tyrosine in crystals of the truncated mutant and makes similar strong polar interactions with the enzyme. The alpha-phosphate group interacts with the main-chain nitrogen of Asp38. The two hydroxyl groups of the ribose form strong interactions with the protein. The 2'-hydroxyl group interacts with the carboxylate of Asp194 and the main-chain nitrogen of Gly192 while the 3'-hydroxyl interacts with a tightly bound water molecule (Wat326). The adenine moiety appears to make no significant polar interactions with the protein. The results of site-directed mutagenesis studies are examined in the light of these refined structures.
The structures of D-xylose isomerase from Arthrobacter strain B3728 containing the polyol inhibitors xylitol and D-sorbitol have been solved at 2.5 A and 2.3 A, respectively. The structures have been refined using restrained least-squares refinement methods. The final crystallographic R-factors for the D-sorbitol (xylitol) bound molecules, for 43,615 (32,989) reflections are 15.6 (14.7). The molecule is a tetramer and the asymmetric unit of the crystal contains a dimer, the final model of which, incorporates a total of 6086 unique protein, inhibitor and magnesium atoms together with 535 bound solvent molecules. Each subunit of the enzyme contains two domains: the main domain is a parallel-stranded alpha-beta barrel, which has been reported in 14 other enzymes. The C-terminal domain is a loop structure consisting of five helical segments and is involved in intermolecular contacts between subunits that make up the tetramer. The structures have been analysed with respect to molecular symmetry, intersubunit contacts, inhibitor binding and active site geometry. The refined model shows the two independent subunits to be similar apart from local deviations due to solvent contacts in the solvent-exposed helices. The enzyme is dependent on a divalent cation for catalytic activity. Two metal ions are required per monomer, and the high-affinity magnesium(II) site has been identified from the structural results presented here. The metal ion is complexed, at the high-affinity site, by four carboxylate side-chains of the conserved residues, Glu180, Glu216, Asp244 and Asp292. The inhibitor polyols are bound in the active site in an extended open chain conformation and complete an octahedral co-ordination shell for the magnesium cation via their oxygen atoms O-2 and O-4. The active site lies in a deep pocket near the C-terminal ends of the beta-strands of the barrel domain and includes residues from a second subunit. The tetrameric molecule can be considered to be a dimer of "active" dimers, the active sites being composed of residues from both subunits. The analysis has revealed the presence of several internal salt-bridges stabilizing the tertiary and quaternary structure. One of these, between Asp23 and Arg139, appears to play a key role in stabilizing the active dimer and is conserved in the known sequences of this enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)
The crystal structure of a deletion mutant of tyrosyl-tRNA synthetase from Bacillus stearothermophilus has been determined at 2.5 A resolution using molecular replacement techniques. The genetically engineered molecule catalyses the activation of tyrosine with kinetic properties similar to those of the wild-type enzyme but no longer binds tRNATyr. It contains 319 residues corresponding to the region of the polypeptide chain for which interpretable electron density is present in crystals of the wild-type enzyme. The partly refined model of the wild-type enzyme was used as a starting point in determining the structure of the truncated mutant. The new crystals are of space group P2(1) and contain the molecular dimer within the asymmetric unit. The refined model has a crystallographic R-factor of 18.7% for all reflections between 8 and 2.5 A. Each subunit contains two structural domains: the alpha/beta domain (residues 1 to 220) containing a six-stranded beta-sheet and the alpha-helical domain (residues 248 to 319) containing five helices. The alpha/beta domains are related by a non-crystallographic dyad while the alpha-helical domains are in slightly different orientations in the two subunits. The tyrosine substrate binds in a slot at the bottom of a deep active site cleft in the middle of the alpha/beta domain. It is surrounded by polar side-chains and water molecules that are involved in an intricate hydrogen bonding network. Both the alpha-amino and hydroxyl groups of the substrate make good hydrogen bonds with the protein. The amino group forms hydrogen bonds with Tyr169-OH, Asp78-OD1 and Gln173-OE1. The phenolic hydroxyl group forms hydrogen bonds with Asp76-OD1 and Tyr34-OH. In contrast, the substrate carboxyl group makes no direct interactions with the enzyme. The results of both substrate inhibition studies and site-directed mutagenesis experiments have been examined in the light of the refined structure.
The C alpha backbones of the glucose isomerase molecules of Streptomyces rubiginosus and Arthrobacter have been determined by X-ray crystallography and compared. Each molecule is a tetramer of eight-stranded alpha/beta barrels, and the mode of association of the tetramers is identical in each case. The Arthrobacter electron density shows four additional amino acids at the carboxyl terminus. There is also an insertion of six amino acids at position 277, and two individual insertions at about positions 348 and 357 (numbering according to the Streptomyces structure). There is a close structural homology throughout the whole molecule, which is most accurate up to position 325. The r.m.s. displacement for 315 homologous C alpha positions up to this position is 0.92 A.
Diffraction data for alpha-chymotrypsin crystals at -10 degrees C were measured at 1.68 A resolution and refined by restrained structure-factor least-squares refinement. The two independent chymotrypsin molecules in the crystallographic asymmetric unit were refined independently. The overall structure of alpha-chymotrypsin is little changed from published co-ordinates. The root-mean-square shift of C alpha co-ordinates is 0.42 A, co-ordinates for the two molecules showing a root-mean-square difference of 0.19 A. Certain regions with high disorder (residues 9 to 14, 73 to 79) remain difficult to interpret and several side-chains are disordered. Some water molecule positions have been changed. The absence of the tosyl group has made a significant difference to the refined structure at the active site. This now agrees closely with other enzymes of the trypsin family that have been refined at high resolution. There is a strong hydrogen bond between N epsilon 2 (His57) and O gamma (Ser195) in the free enzyme, in line with the published description of the charge relay system.
Crystalline complexes of tyrosyl-tRNA synthetase from Bacillus stearothermophilus were prepared with adenosine, AMP, ATP and PPi, all in the presence of tyrosinol, which binds strongly to the tyrosine binding site but cannot be adenylated by ATP. The hydrolysis of ATP in the presence of crystalline tyrosyl-tRNA synthetase (or redissolved crystals) was checked in the absence of tyrosine or with tyrosinol. No ATPase activity due to the enzyme was detected under these conditions. Difference Fourier analysis shows that tyrosinol binds to the tyrosine binding site with the same occupancy as the amino acid. Comparison between tyrosine and tyrosinol shows the location of the extra oxygen atom of the tyrosine carboxylate. Adenosine, AMP and ATP are weakly bound to the enzyme in the presence of tyrosinol. Even when ATP is present at a concentration greater than Km for adenylation, it is not sufficiently strongly bound to give a recognizable density for adenine. However, some significant peaks of density are present near the tyrosine binding site. One of them is at the usual ribose binding site, and may possibly represent ribose binding with a low occupancy. When AMP is bound a similar but not identical arrangement of density is observed.
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The three-dimensional structures of two animoacyl-tRNA synthetases, the methionyl-tRNA synthetase from Escherichia coli (MetRS) and the tyrosyl-tRNA synthetase from Bacillus stearothermophilus (TyrRS), show a remarkable similarity over a span of about 140 amino acids. The region of homologous folding corresponds to a five-stranded parallel beta-sheet, including a mononucleotide-binding fold. One cysteine and two histidine residues that were found to be invariant in the amino acid sequences occupy similar places in the nucleotide-binding fold. In TyrRS, these residues are close to the adenylate binding site, and in MetRS to the Mg2+-ATP binding site.
Oligodeoxynucleotide-directed mutagenesis has been used on the gene of tyrosyl-tRNA synthetase from Bacillus stearothermophilus to produce mutant enzymes altered at the adenosine 5'-triphosphate (ATP) binding site. Deliberate attempts were made to alter rather than destroy enzymic activity so that kinetic measurements may be made to identify the subtle roles of the enzyme-substrate interactions in catalysis. Cys-35, the -SH group of which is involved in binding the 3'-OH of the ribose ring of ATP, has been mutated to a serine residue [Winter, G., Fersht, A. R., Wilkinson, A. J., Zoller, M., & Smith, M. (1982) Nature (London) 299, 756-758] or glycine residue. The mutant enzymes are less active than the wild type, and the reduction in activity can be attributed to a decrease in the value of kcat and an increase in KM. Thus, the interaction energy of the side chain of Cys-35 with the substrate is not fully realized in the enzyme-substrate complex but is used preferentially to stabilize the transition state. Relative to its absence in the Gly-35 mutant, the side chain of Cys-35 is calculated to stabilize the transition state for pyrophosphate exchange by 1.2 kcal/mol and the transition state for aminoacylation by 1.0 kcal/mol.
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There is an increasing body of crystallographic evidence for disorder in parts of the main polypeptide chain of certain proteins. The zymogens of pancreatic serine proteinases and tyrosyl-tRNA synthetase are used as examples. It is suggested that reactivity and specificity are favoured by an appropriate rigid conformation, and that disorder may often be involved in some kind of control function.
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An enzyme is designed to bind most tightly to a substrate when it is in the transition state of the reaction which the enzyme catalyses. The consequent reduction of the activation energy of the reaction constitutes the catalytic mechanism. The energetic contributions of different features of the interaction can only be crudely assessed, but they are dominated by entropically driven effects. The binding site of trypsin orients the substrate so that the reacting groups are correctly placed for reaction to occur. Apart from two side chains which take part in chemical steps of the reaction, the enzyme behaves almost as a rigid body. The full binding interactions are only developed when the substrate is in an intermediate stage of the reaction. The tightly bound complexes of trypsin with protein trypsin inhibitors have proved amenable to structural analysis. Enzyme inhibitor interactions, which account for almost 80 kJ mol-1 of interaction energy, are known fairly accurately. The similarity of the two known trypsin inhibitor structures, close to the primary binding site, indicates a high specificity, even for this simple interaction. In cases where no large conformational changes occur the specificity of an enzyme should be predictable from accurate knowledge of its tertiary structure.