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D Ringe

Publications and source records attributed to D Ringe.

At least 37 records · Page 2Linked to original sources
PubMed1990

The structure of iron superoxide dismutase from Pseudomonas ovalis complexed with the inhibitor azide.

The 2.9 A resolution structure of iron superoxide dismutase (FeSOD) (EC 1.15.1.1) from Pseudomonas ovalis complexed with the inhibitor azide was solved. Comparison of this structure with free enzyme shows that the inhibitor is bound at the open coordination position of the iron, with a bond length of 2.0 A. The metal moves by 0.4 A into the trigonal plane to produce an orthogonal geometry at the iron. Binding of the inhibitor also causes a movement of the axial ligand (histidine 26) away from the metal, a lengthening of the iron-histidine bond, and a rotation of the histidine 74 ring. The inhibitor possesses contacts in the binding pocket with a pair of conserved tryptophan residues and with the side chains of tyrosine 34 and glutamine 70. This glutamine is conserved between all FeSODs, but is absent in MnSOD. Comparisons with MnSOD show that a different glutamine which possesses the same interactions in the active site as Gln70 in FeSOD is conserved at position 154 in the overall SOD sequence, implying that while manganese and FeSODs are structural homologues in a global sense, their functional and evolutionary relationship is that of second-site mutation revertants.

Amino Acid Sequence↗
PubMed1989

Effects of D-serine on bacterial D-amino acid transaminase: accumulation of an intermediate and inactivation of the enzyme.

Incubation of pure bacterial D-amino acid transaminase with D-serine or erythro-beta-hydroxy-DL-aspartic acid, which are relatively poor substrates, leads to generation of a new absorbance band at 493 nm that is probably the quinonoid intermediate. The 420-nm absorbance band (due to the pyridoxal phosphate coenzyme) decreases, and the 338-nm absorbance band (due to the pyridoxamine phosphate or some other form of the coenzyme) increases. A negative Cotton effect at 493 nm in the circular dichroism spectra is also generated. Closely related D amino acids do not lead to generation of this new absorption band, which has a half-life of the order of several hours. Treatment of the enzyme with the good substrate D-alanine leads to a small but detectable amount of the same absorbance band. D-Serine but not erythro-beta-hydroxyaspartate leads to inactivation of D-amino acid transaminase, and D-alanine affords partial protection. The results indicate that D-serine is a unique type of inhibitor in which the initial steps of the half-reaction of transamination are so slow that a quinonoid intermediate with a 493-nm absorption band accumulates. A derivative formed from this intermediate inactivates the enzyme.

Circular Dichroism↗
PubMed1989

Stereospecificity of reactions catalyzed by bacterial D-amino acid transaminase.

The spectral shift from 420 to 338 nm when pure bacterial D-amino acid transaminase binds D-amino acid substrates is also exhibited in part by high concentrations of L-amino acids (L-alanine and L-glutamate) but not by simple dicarboxylic acids or monoamines. Slow processing of L-alanine to D-alanine was observed both by coupled enzymatic assays using D-amino acid oxidase and by high pressure liquid chromatography analysis employing an optically active chromophore (Marfey's reagent). When the acceptor for L-alanine was alpha-ketoglutarate, D-glutamate was also formed. This minor activity of the transaminase involved both homologous (L-alanine and D-alanine) and heterologous (L-alanine and D-glutamate) substrate pairs and was a function of the nature of the keto acid acceptor. In the presence of alpha-ketoisovalerate, DL-alanine was almost completely processed to D-valine; within the limits of the assay no L-valine was detected. With alpha-ketoisocaproate, 90% of the DL-alanine was converted to D-leucine. In the mechanism of this transaminase reaction, there may be more stereoselective constraints for the protonation of the quinonoid intermediate during the second half-reaction of the transamination reaction, i.e. the donation of the amino group from the pyridoxamine 5'-phosphate coenzyme to a second keto acid acceptor, than during removal of the alpha proton in the initial steps of the reaction pathway. Thus, with this D-amino acid transaminase, the discrete steps of transamination ensure fidelity of the stereospecificity of reaction pathway.

Bacillus↗
PubMed1989

2.8-A-resolution crystal structure of an active-site mutant of aspartate aminotransferase from Escherichia coli.

The three-dimensional structure of a mutant of the aspartate aminotransferase from Escherichia coli, in which the active-site lysine has been substituted by alanine (K258A), has been determined at 2.8-A resolution by X-ray diffraction. The mutant enzyme contains pyridoxamine phosphate as cofactor. The structure is compared to that of the mitochondrial aspartate aminotransferase. The most striking differences, aside from the absence of the lysine side chain, occur in the positions of the pyridoxamine group and of tryptophan 140.

Aspartate Aminotransferases↗
PubMed1989

Crystallographic studies of the mechanism of xylose isomerase.

The mechanism of xylose isomerase (EC 5.3.1.5) has been studied with X-ray crystallography. Four refined crystal structures are reported at 3-A resolution: native enzyme, enzyme + glucose, enzyme + glucose + Mg2+, and enzyme + glucose + Mn2+. One of these structures (E.G.Mg) was determined in a crystal mounted in a flow cell. The other structures were equilibrium experiments carried out by soaking crystals in substrate containing solution. These structures and other studies suggest that, contrary to expectation, xylose isomerase may not use the generally expected base-catalyzed enolization mechanism. A mechanism involving a hydride shift is consistent with the structures presented here and warrants further investigation. Additional evidence in support of a hydride shift comes from comparing xylose isomerase with triosephosphate isomerase which is known to catalyze an analogous reaction via an enediol intermediate. Evidence is presented that suggests that aldose-ketose isomerases can be divided into two groups. Phospho sugar isomerases generally do not require a metal ion for activity and show exchange of substrate protons with solvent. In contrast, simple sugar isomerases all require a metal ion and show very low solvent exchange. These observations are rationalized on the basis of the need for stereospecific sugar binding.

Aldose-Ketose Isomerases↗
PubMed1989

Activity and spectroscopic properties of bacterial D-amino acid transaminase after multiple site-directed mutagenesis of a single tryptophan residue.

One of the three tryptophan residues per subunit of thermostable D-amino acid transaminase, Trp-139, is close to the active-site Lys-145 in the sequence of the protein. This tryptophan has been changed to several other types of residues by site-directed mutagenesis. The only mutant protein that was sufficiently active and stable for study had Phe substituted for Trp (W139F). The spectroscopic properties of this mutant enzyme differed from those of the wild-type transaminase. For example, denatured W139F showed the expected decrease in fluorescence emission intensity at 350 nm due to the deletion of one Trp residue, but the fluorescence emission of the wild-type and W139F enzymes in the native state did not differ in intensity. This result suggests that the fluorescence of Trp-139 in the native, wild-type enzyme is not manifested perhaps due to its proximity to the coenzyme, pyridoxal phosphate. Results of energy-transfer studies at several wavelengths could also be interpreted as due to the proximity of Trp-139 and the coenzyme. Circular dichroism studies indicated that the negative Cotton effect at 420 nm due to the coenzyme was still present in W139F. However, the 280-nm optically active band present in the wild-type enzyme was greatly diminished in W139F. The mutant protein with Asp at position 139 (W139D) could not be isolated presumably because it was degraded. The other mutant enzymes, W139P, W139A, and W139H, were isolated with partial activities (15-35%) that were slowly lost upon storage at 4 degrees C. Overall, these results indicate the importance of Trp-139 in the thermostable D-amino acid transaminase.

Bacillus↗
PubMed1989

Charge motion in MbCO crystals after flash photolysis.

Charge motion accompanying the dissociation and recombination of carbon monoxide to oriented myoglobin crystals has been observed. The magnitude of the electrical signals detected after photodissociation by electrodes on either side of MbCO crystals of type A is consistent with the x-ray data showing that the doubly charged iron ion lies in the mean heme plane when a ligand is bound and moves out of the plane when deligated. Beyond 10 ms, the time development of the electrical signal is consistent with the kinetics observed optically after flash photolysis on the same crystals.

Carbon Monoxide↗
PubMed1988

Characterization of crystals of xylose isomerase from Streptomyces violaceoniger.

Crystals of the tetrameric xylose isomerase from Streptomyces violaceoniger have been examined by x-ray analysis. Octahedral crystals with a maximum dimension of 0.7 mm were grown from ammonium sulfate solution. They possess the symmetry of P4(1)2(1)2 or P4(3)2(1)2 space groups, which are crystallographically indistinguishable. The unit cell dimensions are a = b = 140 A and c = 134 A. There is one tetramer of molecular weight 160,000 per asymmetric unit. The crystals diffract to 2.2 A.

Aldose-Ketose Isomerases↗
PubMed1987

Preliminary X-ray data for a D-amino acid amino-transferase from a novel thermophilic Bacillus.

Crystals of the D-amino acid aminotransferase (D-ATA) from a novel thermophilic Bacillus species (Escherichia coli pICT113 cloned gene product) have been examined by X-ray analysis. The crystals grow as hexagonal prisms, with the symmetry of space group P61 or P65 (indistinguishable crystallographically). The cell dimensions are a = b = 135 A, c = 53 A, alpha = beta = 90 degrees, and gamma = 120 degrees. The unit cell has a volume of 850,000 A3 with six asymmetric units per unit cell. There is one dimer of molecular weight 62,000 per asymmetric unit, and the crystals diffract to 2.7 A.

Bacillus↗
PubMed1987

Thermal expansion of a protein.

The thermal expansion of a protein, metmyoglobin, was investigated by analysis of the refined X-ray crystal structures at 80 and 255-300 K. On heating from 80 to 300 K, the volume occupied by myoglobin increases by approximately 3%. The linear thermal expansion coefficient is estimated to be 115 X 10(-6) K-1. This value is more than twice as large as that of liquid water but less than that of benzene. As the temperature is raised, the internal volume change does not come from the large, atom-sized internal cavities in the structure but from an increase in the small, subatomic free volumes between atoms. The largest expansion occurs in the region of the CD and GH corners; both these regions move away from the center of the protein. The remainder of the expansion results from the lengthening of contacts between segments of secondary structure.

Animals↗
PubMed1987

The 3.0 A crystal structure of xylose isomerase from Streptomyces olivochromogenes.

The crystal structure of xylose isomerase [E.C. 5.3.1.5] from Streptomyces olivochromogenes has been determined to 3.0 A resolution. The crystals belong to space group P22(1)2(1) with unit cell parameters a = 98.7, b = 93.9, c = 87.7. The asymmetric unit contains half of a tetrameric molecule of 222 symmetry. The two-fold axis relating the two molecules in the asymmetric unit is close to where a crystallographic two-fold would be if the space group were I222. This causes the diffraction pattern to have strong I222 pseudo-symmetry, so all data were collected in this pseudo-space group. Since the sequence of this enzyme has not been reported, a polyalanine backbone has been fitted to the electron density. Xylose isomerase has two domains: the N-terminal domain is an eight-stranded alpha/beta barrel of 299 residues. The C-terminal domain is a large loop of 50 residues which is involved in intermolecular contacts. Comparison of xylose isomerase with the archetypical alpha/beta barrel protein, triose phosphate isomerase, reveals that the proteins overlap best when the third (alpha beta) strand of xylose isomerase is superimposed on the first (alpha beta) strand of triose phosphate isomerase. This same overlap has also been found between the muconate lactonising enzyme and triose phosphate isomerase [Goldman et al. (1987) J. Mol. Biol., in press].

Aldose-Ketose Isomerases↗
PubMed1986

X-ray diffraction analysis of the inactivation of chymotrypsin by 3-benzyl-6-chloro-2-pyrone.

The inactivation of chymotrypsin by 3-benzyl-6-chloro-2-pyrone has been studied. A covalent adduct is formed that deacylates slowly with a half-life of 23 h. X-ray diffraction analysis at 1.9-A resolution of the inactivator-enzyme complex shows that the gamma-oxygen of the active-site serine (serine-195) is covalently attached to C-1 of (Z)-2-benzylpentenedioic acid, the benzyl group of the inactivator is held in the hydrophobic specificity pocket of the enzyme, and the free carboxylate forms a salt bridge with the active-site histidine (histidine-57). The conformational changes that occur in the protein as a result of complexation are described. It is proposed that formation of the salt bridge prevents access of water and, therefore, hydrolysis of the acyl-enzyme.

Binding Sites↗
PubMed1986

Preliminary X-ray data for aspartate aminotransferase from Escherichia coli.

Crystals of the aspartate aminotransferase from Escherichia coli (aspC gene product) have been examined by X-ray analysis. The crystals grow as elongated rectangular prisms, with the symmetry of space group C2221. Unit cell dimensions are a = 156 A, b = 87.6 A, c = 80.6 A and alpha = beta = gamma = 90 degrees. There is one protein subunit of molecular weight 43,600 per asymmetric unit.

Aspartate Aminotransferases↗
PubMed1986

Study of protein dynamics by X-ray diffraction.

Properly carried out, high-resolution X-ray diffraction data collection followed by careful least-squares refinement can give the spatial distribution of the high-frequency mean-square displacements in a protein. These displacements reflect both individual atomic fluctuations in hard variables (bond lengths and bond angles) and collective motions involving soft variables (torsion angles, nonbonded interactions). Lower frequency, large amplitude motions and rapid but improbable motions are not quantifiable, but they may lead to such complete disorder that their existence can at least be inferred from the absence of interpretable electron density for some sections of the structure. Interior residues are more rigid than groups on the surface, and structural constraints are reflected in restricted motion even for surface residues. Amplitudes of motion of 0.5 A or greater are not uncommon. The temperature dependence of these fast motions varies considerably over the structure. In general, large [chi 2] values have large temperature dependence, while small displacements are less affected by temperature; however, exceptions are common. Significant reduction in [chi 2] on cooling establishes that proteins are mobile even in the crystalline state, and that static disorder is not the dominant contributor to the individual mean square displacements. Disordered regions in electron density maps are no longer automatically taken as signs of errors in structure determination. It is now recognized that the absence of strong electron density is often an indicator of conformational flexibility. Some of the functional roles for protein dynamics are beginning to be understood. Missing from these results are the physicochemical details that can be extracted from thermal motion analysis of small molecule crystal structures. Application of these methods to protein data is very difficult, but it is well to remember that just over 10 years ago it was commonly felt that protein structures could not even be refined. Certainly some small, well-diffracting proteins should be amenable to many of the sophisticated small-molecule analyses, as they yield X-ray data to resolutions comparable to simple organic structures. The most important type of analysis that awaits is anisotropic B factor refinement, which would give the principal directions of motion added to the amplitude information now obtained. Unfortunately, refinement of unrestrained anisotropic thermal elipsoids requires six parameters for each atom instead of a single isotropic B parameter, and even 1.5 A resolution data do not provide enough overdeterminacy.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Inactivation of chymotrypsin by 5-benzyl-6-chloro-2-pyrone: 13C NMR and X-ray diffraction analyses of the inactivator-enzyme complex.

The inactivation of chymotrypsin by 5-benzyl-6-chloro-2-pyrone has been studied. Chloride analysis of the inactivated enzyme suggests that chlorine is no longer present in the complex. 13C NMR spectroscopy of chymotrypsin inactivated with 5-benzyl-6-chloro-2-pyrone-2,6-13 C2 shows the presence of two new resonances from the protein-bound inactivator. The chemical shift values of these resonances are consistent with an intact pyrone ring on the enzyme as well as the replacement of the C-6 chlorine by a different heteroatom. X-ray diffraction analysis at 1.5-A resolution of the inactivator-enzyme complex demonstrates that the gamma-oxygen of the active site serine residue (serine 195) is covalently attached to C-6 of the inactivator and that the pyrone ring is intact. The 5-benzyl group of the inactivator is bound to the enzyme in the hydrophobic specificity pocket. The conformational changes that occur in the protein as a result of complexation with the inactivator are discussed.

Binding Sites↗