Biomedical subjects

D Ringe

Publications and source records attributed to D Ringe.

At least 55 records · Page 3Linked to original sources

PubMed1996

The structural basis for pseudoreversion of the H95N lesion by the secondary S96P mutation in triosephosphate isomerase.

The structural basis for the 3000-fold decrease in catalytic efficiency of the H95N mutant chicken triosephosphate isomerase and the 60-fold regain of catalytic efficiency in the double mutant, H95N.S96P, have been analyzed. The results from a combination of X-ray crystallography and Fourier transform infrared spectroscopy experiments indicate that the predominant defect in the H95N mutant isomerase appears to be its inability to bind the substrate in a coplanar, cis conformation. The structures of each mutant isomerase were determined from X-ray crystallography of the complex of phosphoglycolohydroxamate (PGH), an intermediate analog with the isomerase, and each was solved to a resolution of 1.9 A. The PGH appeared to be in two different conformations in which the enediol-mimicking atoms, O2-N2-C1-O1, of the PGH were not coplanar. No density was observed that would correspond to the coplanar conformation. Two bands are observed for the dihydroxyacetone phosphate carbonyl in the H95N mutant FTIR spectrum, and these can be explained if the O1 of DHAP, like the O1 of PGH in the crystal structure, is in two different positions. Two ordered water molecules are located between O1 of PGH and N delta of N95. Comparison of the structure of the pseudorevertant, H95N.S96P with that for the H95N single mutant, shows that S96P mutation causes the double mutant to regain the ability to bind PGH predominantly in the coplanar, cis conformation. Electron density for a single ordered water molecule bridging the N95 amide side chain and the O2 of PGH is observed, but the density was weak, perhaps indicating that the water molecule is somewhat disordered. Whether or not a water molecule is hydrogen bonded to O2 of PGH may explain the two carbonyl stretching frequencies observed for the GAP carbonyl. Together, the crystal structures and the FTIR data allow a complete explanation of the catalytic properties of these two mutant isomerases.

Crystallography, X-Ray↗

PubMed1996

Interaction of pyridoxal 5'-phosphate with tryptophan-139 at the subunit interface of dimeric D-amino acid transaminase.

The crystal structure of dimeric bacterial D-amino acid transaminase shows that the indole rings of the two Trp-139 side chains face each other in the subunit interface about 10 angstroms from the coenzyme, pyridoxal 5'-phosphate. To determine whether it has a role in the catalytic efficiency of the enzyme or interacts with the coenzyme, Trp-139 has been substituted by several different types of amino acids, and the properties of these recombinant mutant enzymes have been compared to the wild-type enzyme. In the native wild-type holoenzyme, the fluorescence of one of the three Trp residues per monomer is almost completely quenched, probably due to its interaction with PLP since in the native wild-type apoenzyme devoid of PLP, tryptophan fluorescence is not quenched. Upon reconstitution of this apoenzyme with PLP, the tryptophan fluorescence is quenched to about the same extent as it is in the native wild-type enzyme. The site of fluorescence quenching is Trp-139 since the W139F mutant in which Trp-139 is replaced by Phe has about the same amount of fluorescence as the wild-type enzyme. The circular dichroism spectra of the holo and the apo forms of both the wild-type and the W139F enzymes in the far-ultraviolet show about the same degree of ellipticity, consistent with the absence of extensive global changes in protein structure. Furthermore, comparison of the circular dichroism spectrum of the W139F enzyme at 280 nm with the corresponding spectral region of the wild-type enzyme suggests a restricted microenvironment for Trp-139 in the latter enzyme. The functional importance of Trp-139 is also demonstrated by the finding that its replacement by Phe, His, Pro, or Ala gives mutant enzymes that are optimally active at temperatures below that of the wild-type enzyme and undergo the E-PLP --> E-PMP transition as a function of D-Ala concentration with reduced efficiency. The results suggest that a fully functional dimeric interface with the two juxtaposed indole rings of Trp-139 is important for optimal catalytic function and maximum thermostability of the enzyme and, furthermore, that there might be energy transfer between Trp-139 and coenzyme PLP.

Catalysis↗

PubMed1996

The ubiquitous cofactor NADH protects against substrate-induced inhibition of a pyridoxal enzyme.

In the usual reaction catalyzed by D-amino acid transaminase, cleavage of the alpha-H bond is followed by the reversible transfer of the alpha-NH2 to a keto acid cosubstrate in a two-step reaction mediated by the two vitamin B6 forms pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP). We report here a reaction not on the main pathway, i.e., beta-decarboxylation of D-aspartate to D-alanine, which occurs at 0.01% the rate of the major transaminase reaction. In this reaction, beta-C-C bond cleavage of the single substrate D-aspartate occurs rather than the usual alpha-bond cleavage in the transaminase reaction. The D-alanine produced from D-aspartate slowly inhibits both transaminase and decarboxylase activities, but NADH or NADPH instantaneously prevent D-aspartate turnover and D-alanine formation, thereby protecting the enzyme against inhibition. NADH has no effect on the enzyme spectrum itself in the absence of substrates, but it acts on the enzyme.D-aspartate complex with an apparent dissociation constant of 16 microM. Equivalent concentrations of NAD or thiols have no such effect. The suppression of beta-decarboxylase activity by NADH occurs concomitant with a reduction in the 415-nm absorbance due to the PLP form of the enzyme and an increase at 330 nm due to the PMP form of the enzyme. alpha-Ketoglutarate reverses the spectral changes caused by NADH and regenerates the active PLP form of the enzyme from the PMP form with an equilibrium constant of 10 microM. In addition to its known role in shuttling electrons in oxidation-reduction reactions, the niacin derivative NADH may also function by preventing aberrant damaging reactions for some enzyme-substrate intermediates. The D-aspartate-induced effect of NADH may indicate a slow transition between protein conformational studies if the reaction catalyzed is also slow.

Amino Acids↗

PubMed1996

Locating and characterizing binding sites on proteins.

This review article begins with a discussion of fundamental differences between substrates and inhibitors, and some of the assumptions and goals underlying the design of a new ligand to a target protein. An overview is given of the methods currently used to locate and characterize ligand binding sites on protein surfaces, with focus on a novel approach: multiple solvent crystal structures (MSCS). In this method, the X-ray crystal structure of the target protein is solved in a variety of organic solvents. Each type of solvent molecule serves as a probe for complementary binding sites on the protein. The probe distribution on the protein surface allows the location of binding sites and the characterization of the potential ligand interactions within these sites. General aspects of the application of the MSCS method to porcine pancreatic elastase is discussed, and comparison of the results with those from X-ray crystal structures of elastase/inhibitor complexes is used to illustrate the potential of the method in aiding the process of rational drug design.

Animals↗

PubMed1996

Identification of the primary metal ion-activation sites of the diphtheria tox repressor by X-ray crystallography and site-directed mutational analysis.

The diphtheria tox repressor, DtxR, is a 226 amino acid transition metal ion-activated regulatory protein that controls the expression of diphtheria toxin in toxigenic Corynebacterium diphtheriae. The previously solved three-dimensional DtxR structures have identified two potential metal ion binding sites which may play a role in the activation of DNA binding by the repressor. We have used both X-ray crystallographic and site-directed mutational analysis of DtxR(C102D)-Ni2+ complexes and DtxR to identify the metal ion-binding site which results in the activation of the repressor. We demonstrate that DtxR contains both a primary and an ancillary metal ion binding site. The primary site functions directly in the activation of DNA binding. In contrast, the ancillary site contributes weakly, if at all, to activation.

Bacterial Proteins↗

PubMed1995

The structural basis for pseudoreversion of the E165D lesion by the secondary S96P mutation in triosephosphate isomerase depends on the positions of active site water molecules.

The structural basis for the improvement in catalytic efficiency of the mutant E165D chicken triosephosphate isomerase by the secondary mutation, S96P, has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. All X-ray structures were of the complex of phosphoglycolohydroxamate (PGH), an intermediate analog, with the isomerase, and each was solved to a resolution of 1.9 A. Comparison of the structure of the double mutant, E165D.S96P, with that of the single mutant, E165D, as well as with the wild-type isomerase shows only insignificant differences in the positions of the side chains in all of the mutants when compared with the wild-type isomerase, except that in both the E165D and E165D.S96P mutants, the aspartate side chain was approximately 0.7 A further away from the substrate analog than the glutamate side chain. Significant differences were observed in the crystal structure of the E165D.S96P double mutant in the positions of ordered water molecules bound at the active site. The loss of two water molecules located near the side chain at position 165 was observed in isomerases containing the S96P mutation. The resulting increase in hydrophobicity of the pocket probably causes an increase in the pKa of the catalytic base, D165, thereby improving its basicity. A new ordered water molecule was observed underneath the bound PGH in the E165D.S96P structure, which likely decreases the pKa's of the substrate protons, thereby increasing their acidity. An enzyme derived carbonyl stretch at 1746 cm-1 that is only observed in the IR spectrum of the E165D.S96P double mutant isomerase with bound substrates has been assigned to a stable ground state protonated D165-enediol(ate) intermediate complex. Thus, the gain in activity resulting from the S96P second site change probably results from a combination of improving the basicity of the enzyme, improving the acidity of the substrate protons, and stabilization of a reaction intermediate. All three of these effects seem to be caused by changes in bound water molecules.

Animals↗

PubMed1995

Revision of the amino-acid sequence of 3-isopropylmalate dehydrogenase from Salmonella typhimurium by means of X-ray crystallography.

The amino acid (aa) sequence of the leuB gene product of Salmonella typhimurium, 3-isopropylmalate dehydrogenase (IPMDH), has been revised using electron density maps from X-ray structure determination. The nucleotide (nt) sequence of both strands of leuB has been redetermined to confirm the crystallographic findings. It does not agree with the previously reported S. typhimurium leuB nucleotide sequence [Andreadis and Rosenthal, Biochim. Biophys. Acta 1129 (1992) 228-230].

3-Isopropylmalate Dehydrogenase↗

PubMed1995

Structures of the apo- and the metal ion-activated forms of the diphtheria tox repressor from Corynebacterium diphtheriae.

The diphtheria tox repressor (DtxR) of Corynebacterium diphtheriae plays a critical role in the regulation of diphtheria toxin expression and the control of other iron-sensitive genes. The crystal structures of apo-DtxR and of the metal ion-activated form of the repressor have been solved and used to identify motifs involved in DNA and metal ion binding. Residues involved in binding of the activated repressor to the diphtheria tox operator, glutamine 43, arginine 47, and arginine 50, were located and confirmed by site-directed mutagenesis. Previous biochemical and genetic data can be explained in terms of these structures. Conformational differences between apo- and Ni-DtxR are discussed with regard to the mechanism of action of this repressor.

Amino Acid Sequence↗

PubMed1995

Crystal structure of a D-amino acid aminotransferase: how the protein controls stereoselectivity.

The three-dimensional structure of D-amino acid aminotransferase (D-AAT) in the pyridoxamine phosphate form has been determined crystallographically. The fold of this pyridoxal phosphate (PLP)-containing enzyme is completely different from those of any of the other enzymes that utilize PLP as part of their mechanism and whose structures are known. However, there are some striking similarities between the active sites of D-AAT and the corresponding enzyme that transaminates L-amino acids, L-aspartate aminotransferase. These similarities represent convergent evolution to a common solution of the problem of enforcing transamination chemistry on the PLP cofactor. Implications of these similarities are discussed in terms of their possible roles in the stabilization of intermediates of a transamination reaction. In addition, sequence similarity between D-AAT and branched chain L-amino acid aminotransferase suggests that this latter enzyme will also have a fold similar to that of D-AAT.

Alanine Transaminase↗

PubMed1995

Interaction of a peptidomimetic aminimide inhibitor with elastase.

The crystal structure of an aminimide analog of a dipeptide inhibitor of porcine pancreatic elastase bound to its target serine protease has been solved. The peptidomimetic molecule binds in the same fashion as the class of dipeptides from which it was derived, making similar interactions with the subsites on the elastase surface. Because aminimides are readily synthesized from a wide variety of starting materials, they form the basis for a combinatorial chemistry approach to rational drug design.

Amino Acid Sequence↗

PubMed1995

Nature of the inactivation of elastase by N-peptidyl-O-aroyl hydroxylamine as a function of pH.

The mechanism of inactivation of porcine pancreatic elastase (PPE) by N-peptidyl-O-aroylhydroxylamine was studied by X-ray crystallography. The inactivator forms a stable complex with the enzyme by means of a covalent attachment to the active site Ser 203(195) O gamma. The nature of the complex is, however, different depending on the pH at which the inactivation reaction occurs. At pH 5, the complex formed is a hydroxylamine derivative of Ser 203(195) in which the O gamma of serine is the oxygen of the hydroxylamine derivative. At pH 7.5, the complex formed is a carbamate derivative at Ser 203(195) O gamma. In both types of complexes, the inactivator binds in the S' subsites of the enzyme instead of forming the usual antiparallel beta-sheet with the S subsites. The implication for the mechanism of inactivation at different pHs is discussed.

Animals↗

PubMed1995

Design, synthesis, and characterization of a potent xylose isomerase inhibitor, D-threonohydroxamic acid, and high-resolution X-ray crystallographic structure of the enzyme-inhibitor complex.

The binding of a potent inhibitor to the enzyme D-xylose isomerase from Streptomyces olivochromogenes was examined by kinetics and X-ray crystallography. The inhibitor D-threonohydroxamic acid (THA) was designed to mimic the putative transition state of the isomerization step catalyzed by the enzyme on the substrate xylose. THA was synthesized and found to be a slow-binding competitive inhibitor with the substrate glucose. The Ki < or = 100 nM was at least one million-fold less than the KM for glucose. The X-ray crystallographic structure of xylose isomerase with THA soaked into the crystals (concentration = 1000Ki) was obtained to 1.6-A resolution and refined to an R factor of 21.6%. The free enzyme and the enzyme in the xylose isomerase-THA complex show no significant structural differences. THA binds in an analogous fashion to glucose, in a linear conformation, forming ligands with Mg-1 and Mg-2 and hydrogen bonds with His53 and Lys182. On the basis of these similarities to glucose binding and its potent inhibition, we propose that THA resembles the transition state for the enzyme-catalyzed hydride transfer reaction. The THA C2 hydroxyl forms a bridging ligand between Mg-1 and Mg-2; it must be deprotonated to do so. By analogy, we propose that, during the catalytic reaction, C2 of the substrate glucose is deprotonated, and that this proton can be moved to the C1 hydroxyl concomitant with hydride transfer. We find evidence for metal movement during catalysis upon deprotonation of the C2 hydroxyl, to allow formation of a bridging ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldose-Ketose Isomerases↗

PubMed1995

Structural analysis of the active site of porcine pancreatic elastase based on the X-ray crystal structures of complexes with trifluoroacetyl-dipeptide-anilide inhibitors.

The X-ray crystal structures of two new (trifluoroacetyl)dipeptide p-(trifluoromethyl)anilide (TFA-dipeptide-TFM) inhibitors complexed to porcine pancreatic elastase are presented. TFA-Val-Ala-TFM and TFA-Phe-Ala-TFM both bind to elastase with the TFA group in the S1 subsite, Val or Phe in the S2 subsite, Ala in the S3 subsite, and the TFM group in the S4 subsite. Five other TFA-dipeptide-anilide/elastase crystal structures are available (two TFA-X-Ala-p-(trifluoromethyl)anilide, X = Lys, Leu, and three TFA-Lys-X-p-isopropylanilide, X = Pro, Leu, Phe). The four inhibitors with the trifluoromethyl substituent on the anilide ring bind in a single mode to elastase, whereas superposition of the three inhibitors with the isopropyl substituent on the anilide ring show three different modes of binding to the protein [Mattos, C., et al. (1994) Nature Struct. Biol. 1, 55-58]. The seven structures are taken together in a detailed analysis of the active site of porcine pancreatic elastase. The inhibition constants for the inhibitors are used in combination with the crystal structures to understand the specificity of the different elastase subsites.

Animals↗

PubMed1995

A functionally diverse enzyme superfamily that abstracts the alpha protons of carboxylic acids.

Mandelate racemase and muconate lactonizing enzyme are structurally homologous but catalyze different reactions, each initiated by proton abstraction from carbon. The structural similarity to mandelate racemase of a previously unidentified gene product was used to deduce its function as a galactonate dehydratase. In this enzyme superfamily that has evolved to catalyze proton abstraction from carbon, three variations of homologous active site architectures are now represented: lysine and histidine bases in the active site of mandelate racemase, only a lysine base in the active site of muconate lactonizing enzyme, and only a histidine base in the active site of galactonate dehydratase. This discovery supports the hypothesis that new enzymatic activities evolve by recruitment of a protein catalyzing the same type of chemical reaction.

Amino Acid Sequence↗

PubMed1995

Purification and crystallization of benzoylformate decarboxylase.

A new large-scale purification method for benzoylformate decarboxylase from Pseudomonas putida has allowed us to undertake an X-ray crystallographic study of the enzyme. The previously observed instability of the enzyme was overcome by addition of 100 microM thiamine pyrophosphate to buffers used in the purification. The final enzyme preparation was more than 97% pure, as determined by denaturing gel electrophoresis and densitometry. The mobility of the enzyme on a gel filtration column indicates that it is a tetramer of 57-kDa subunits. Large, single crystals of benzoylformate decarboxylase were grown from solutions of buffered polyethylene glycol 400, pH 8.5. The crystals diffract to beyond 1.6 A resolution and are stable for days to X-ray radiation. Analysis of X-ray data from the crystals, along with the newly determined quaternary structure, identifies the space group as I222. The unit cell dimensions are a = 82 A, b = 97 A, c = 138 A. An average Vm value for the crystals is consistent with one subunit per asymmetric unit. The subunits of the tetramer must be arranged with tetrahedral 222 symmetry.

Carboxy-Lyases↗

PubMed1995

Catalytic ability and stability of two recombinant mutants of D-amino acid transaminase involved in coenzyme binding.

Of the major amino acid side chains that anchor pyridoxal 5'-phosphate at the coenzyme binding site of bacterial D-amino acid transaminase, two have been substituted using site-directed mutagenesis. Thus, Ser-180 was changed to an Ala (S180A) with little effect on enzyme activity, but replacement of Tyr-31 by Gln (Y31Q) led to 99% loss of activity. Titration of SH groups of the native Y31Q enzyme with DTNB proceeded much faster and to a greater extent than the corresponding titration for the native wild-type and S180A mutant enzymes. The stability of each mutant to denaturing agents such as urea or guanidine was similar, i.e., in their PLP forms, S180A and Y31Q lost 50% of their activities at a 5-15% lower concentration of urea or guanidine than did the wild-type enzyme. Upon removal of denaturing agent, significant activity was restored in the absence of added pyridoxal 5'-phosphate, but addition of thiols was required. In spite of its low activity, Y31Q was able to form the PMP form of the enzyme just as readily as the wild-type and the S180A enzymes in the presence of normal D-amino acid substrates. However, beta-chloro-D-alanine was a much better substrate and inactivator of the Y31Q enzyme than it was for the wild-type or S180A enzymes, most likely because the Y31Q mutant formed the pyridoxamine 5-phosphate form more rapidly than the other two enzymes. The stereochemical fidelity of the Y31Q recombinant mutant enzyme was much less than that of the S180A and wild-type enzymes because racemase activity, i.e., conversion of L-alanine to D-alanine, was higher than for the wild-type or S180A mutant enzymes, perhaps because the coenzyme has more flexibility in this mutant enzyme.

Alanine↗

PubMed1995

What makes a binding site a binding site?

Organic probe molecules have recently been used to define hydrophobic binding sites on the surface of proteins. It appears that the presence of water on the surface of a protein plays a crucial role in the interaction between that protein and its binding site.

Binding Sites↗

PubMed1995

Structure-aided drug design: crystallography and computational approaches.

Ideally, a drug that effectively attacks a specific target will be efficacious with few or no side effects. Historically, drug development has relied on a structure-activity sequential (trial-and-error) approach, testing many compounds and observing their pharmacologic effects. More recently, molecular biologists have investigated a new approach to drug design: starting with a specific target, determining what types of molecules would have a desired effect on that target and then constructing molecules that fit those criteria. Structure-aided drug design is a tool used in this new approach, which involves using crystallographic data and computer processing to describe a molecule precisely--in terms of all binding, Van der Waal's and electrostatic interactions--as a computational entity. The goal of this computational approach is to determine whether a molecule will bind to a target molecule and the character of the ligand interaction.

Computer Simulation↗