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Biomedical subjects

G A Petsko

Publications and source records attributed to G A Petsko.

At least 73 records · Page 4Linked to original sources
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

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

Mechanism of the reaction catalyzed by mandelate racemase: importance of electrophilic catalysis by glutamic acid 317.

In the high-resolution X-ray structure of mandelate racemase (MR) with the competitive inhibitor (S)-atrolactate bound in the active site [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., & Petsko, G. A. (1994) Biochemistry 33, 635-643], the carboxylic acid group of Glu 317 is hydrogen-bonded to the carboxylate group of the bound inhibitor. This geometry suggests that the carboxylic acid functional group of Glu 317 participates as a general acid catalyst in the concerted general acid-general base catalyzed formation of a stabilized enolic tautomer of mandelic acid as a reaction intermediate. To test this hypothesis, the E317Q mutant of MR was constructed and subjected to high-resolution X-ray structural analysis in the presence of (S)-atrolactate. No conformational alterations were observed to accompany the E317Q substitution at 2.1 A resolution. The values for kcat were reduced 4.5 x 10(3)-fold for (R)-mandelate and 2.9 x 10(4)-fold for (S)-mandelate; the values for kcat/Km were reduced 3 x 10(4)-fold. The substrate and solvent deuterium isotope effects measured for both wild-type MR and the E317Q mutant are not multiplicative when deuteriated substrate is studied in D2O, which suggests that the reactions catalyzed by both enzymes are stepwise and involve the formation of stabilized enolic intermediates. In contrast to wild-type MR, E317Q does not catalyze detectable elimination of bromide ion from either enantiomer of p-(bromomethyl)mandelate. However, E317Q is irreversibly inactivated by racemic alpha-phenylglycidate at a rate comparable to that measured for wild-type MR. Taken together, these mechanistic properties confirm the importance of Glu 317 as a general acid catalyst in the reaction catalyzed by wild-type MR. The kcat for wild-type MR and the reduction in kcat observed for E317O are discussed in terms of the analysis recently described by Gerlt and Gassman for understanding the rates and mechanisms of enzyme-catalyzed proton abstraction reactions from carbon acids [Gerlt, J. A., & Gassman, P. G. (1993) J. Am. Chem. Soc. 115, 11552-11568; Gerlt, J. A., & Gassman, P. G. (1993) Biochemistry 32, 11943-11952].

Base Sequence↗
PubMed1995

Mechanism of the reaction catalyzed by mandelate racemase: structure and mechanistic properties of the K166R mutant.

On the basis of the available high-resolution structures of mandelate racemase (MR) from Pseudomonas putida [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., & Petsko, G. A. (1994) Biochemistry 33, 635-643], Lys 166 and His 297 are positioned appropriately to participate in catalysis as acid/base catalysts that either abstract the alpha-proton from the enantiomers of mandelate to form an enolic intermediate or protonate the enolic intermediate to form the enantiomers of mandelate, with Lys 166 participating as the (S)-specific acid/base catalyst and His 297 participating as the (R)-specific acid/base catalyst. In this paper we report the structural and mechanistic properties of the mutant in which Lys 166 has been replaced with arginine (K166R). The structure of K166R has been determined at 1.85 A resolution with the substrate (S)-mandelate bound in the active site. The structure of this complex reveals no geometric alterations in the active site, with the exception that the longer side chain of Arg 166 is necessarily displaced upward from the position occupied by Lys 166 by steric interactions with the bound substrate. In contrast to the H297N mutant of MR [Landro, J. A., Kallarakal, A. T., Ransom, S. C., Gerlt, J. A., Kozarich, J. W., Neidhart, D. J., & Kenyon, G. L. (1991) Biochemistry 30, 9275-9281], the K166R exhibits low levels of racemase activity [kcat is reduced 5 x 10(3)-fold in the (R)- to (S)-direction and 1 x 10(3)-fold in the (S)- to (R)-direction]. The substrate and solvent deuterium isotope effects support a reaction coordinate for the K166R-catalyzed reaction in which the transition state for interconversion of bound (S)-mandelate and the stabilized enolic intermediate is higher in energy that the transition state for interconversion of bound (R)-mandelate and the stabilized enolic intermediate. The solvent deuterium isotope effect when (S)-mandelate is substrate (2.2 +/- 0.3) supports the proposal that the formation of the enolic intermediate involves partial transfer of a solvent-derived proton from Glu 317 to the substrate as the alpha-proton is abstracted [Mitra, B., Kallarakal, A. T., Kozarich, J. W., Gerlt, J. A., Clifton, J. G., Petsko, G. A., & Kenyon, G. L. (1995) Biochemistry 34, 2777-2787].(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
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

Use of a minimum perturbation approach to predict TIM mutant structures.

A minimum perturbation conformational search approach is used to model the structures of the yeast triosephosphate isomerase (TIM) single mutant in which the catalytic base Glu165 is changed to Asp, and the double mutant in which Glu165 is changed to Asp and Ser96 to Pro. In chicken TIM this double mutant is referred to as a pseudo-revertant because some of the catalytic activity lost due to the first mutation is regained when the second mutation occurs. Three minimum energy structures were calculated for the Asp165 conformation in the yeast TIM single mutant and another three for the double mutant. One of the calculated minimum energy conformations for Asp165 in the E165D structure agrees well with the X-ray structure. However, this conformation is not that of the lowest energy and is not one of the three most common conformers for Asp found by Ponder and Richards. This suggests that when an amino acid is introduced it may not be able to conform to the more general rules that apply to protein structures of evolutionary origin. While the van der Waals energy largely determines the allowed minima, the relative ranking of the final minima is determined by electrostatic effects and can therefore be affected by the inclusion of crystal waters in the calculation. When the E165D calculation is repeated with an active-site water molecule fixed in its E165D X-ray structure position, the relative ranking of the minima shifts and the X-ray conformation for Asp165 is the lowest interaction energy conformer. Two of the E165D calculated minimum energy structures are essentially identical to two of the S96P/E165D minima. All of the calculated minima for both the E165D and S96P/E165D mutants position the Asp side chain such that the anti-orbital, and not the more basic syn-orbital, of the carboxylate would be utilized for proton abstraction. This observation may explain why the chicken TIM S96P/E165D mutant, for which the X-ray structure indicates that the syn-orbital is used, is a pseudo-revertant while the yeast TIM double mutant is not; no X-ray structure is available for the latter. The multiplicity of minima found in the present analysis makes clear that predicting the exact orientation of a single side chain is not as simple as might be expected.

Amino Acid Sequence↗
PubMed1995

Composition analysis of alpha-helices in thermophilic organisms.

We present a statistical comparison of the amino acid composition in a secondary structure element, the alpha-helix, of proteins stable at high temperatures with those which are less so. This study has shown that the temperature-dependent Zimm-Bragg helix propagation value s is not a good predictor for the helix-forming tendency of an amino acid in thermostable proteins. However, we have shown that delta s, the change in s from 20 to 60 degrees C, accurately predicts the direction of the probability shift for 15 amino acids in thermostable protein alpha-helices, although it does not predict the magnitude of that change. The residues tyrosine, glycine and glutamine show a significant increase in residency in alpha-helices for thermostable proteins over their non-thermostable counterparts. Significant decreases in alpha-helix residency occur for the residues valine, glutamic acid, histidine, cysteine and aspartic acid in proteins from thermophilic organisms. Aromatic interactions, hydrogen bonding and a reduction of charge may explain the increase observed for tyrosine and glutamine and the decrease in glutamic acid and aspartic acid, although packing considerations cannot be ruled out. The only physical explanation for the increase in glycine would seem to be its positive delta s value.

Amino Acids↗
PubMed1994

Crystallization and preliminary X-ray studies of the diphtheria Tox repressor from Corynebacterium diphtheriae.

Crystals of the diphtheria tox repressor (DtxR) from Corynebacterium diphtheriae suitable for structure determination have been obtained. DtxR activated with transition metal ions represses the expression of the structural gene for the diphtheria toxin, tox, which is encoded on the genome of a family of closely related corynebacteriophages. The space group of the obtained crystals is trigonal P3(1)21 or its enantiomorph P3(2)21 with a = b = 64.2 A, c = 220.5 A, alpha = beta = 90 degrees, gamma = 120 degrees. Two monomers comprise the asymmetric unit. The crystals diffract to a resolution of better than 3 A.

Bacterial Proteins↗
PubMed1994

Expression, crystallization and preliminary crystallographic analysis of human carbonyl reductase.

The cDNA of human placental carbonyl reductase (EC 1.1.1.184), a member of the short-chain dehydrogenase family of enzymes, was introduced into the plasmid vector pET-11a and the enzyme overexpressed in Escherichia coli. Recombinant carbonyl reductase was purified to homogeneity, characterized physically and kinetically, and crystallized for X-ray diffraction study. The recombinant protein was indistinguishable from human tissue carbonyl reductase (CR8.5 form) on the basis of partial sequence analysis, substrate specificity, susceptibility to inhibitors and immunochemical analysis. Similar to the tissue enzyme which which occurs in multiple molecular forms thought to arise from autocatalytic modification by 2-oxocarboxylic acids, a second form of the recombinant enzyme was generated under bacterial growth conditions producing high pyruvate concentrations. Purified recombinant protein, which corresponds to the smallest, most basic tissue form (CR8.5), was crystallized against 20% polyethyleneglycol 6000 in 25 mM 2-(N-morpholino)ethanesulfonic acid buffer (Mes) at pH 6.0 using the hanging drop method. Crystals of human carbonyl reductase diffract to better than 3.0 A, and the diffraction symmetry is consistent with a crystal that belongs to the tetragonal space group P4(1)(3)2(1)2 with unit cell dimensions of a = b = 55 A, c = 175 A, alpha = beta = gamma = 90.0. The asymmetric unit contains one molecule of 30.2 kDa.

Alcohol Oxidoreductases↗
PubMed1994

Direct structural observation of an acyl-enzyme intermediate in the hydrolysis of an ester substrate by elastase.

The method of X-ray crystallographic cryoenzymology has been used to determine the crystal structure of a kinetically significant species on the reaction pathway of a crystalline enzyme. The structure of a specific acyl-enzyme intermediate in the elastase-catalyzed hydrolysis of the N-carbobenzoxy-L-alanine p-nitrophenyl ester has been determined and refined against X-ray diffraction data at 2.3-A resolution. The difference Fourier electron density map clearly shows electron density for the trapped acyl-enzyme. The acyl-enzyme was formed at -26 degrees C and was stabilized at -55 degrees C during data collection, taking advantage of the glass transition in protein dynamics that occurs at around -50 degrees C.

Alanine↗
PubMed1994

Analysis of two-residue turns in proteins.

The conformational properties of tight two-residue beta-turns in proteins are examined by empirical energy function calculations. Twenty-five tight turns are studied in isolation, in the presence of the protein, and in the presence of the protein and crystal water molecules. The conformational properties are subdivided into those that are intrinsic to the turn and those that depend on the protein and water environment. Two factors are shown to determine the conformation of a tight beta-turn. One is the twist of the beta-sheet (responsible for selecting either a type I' or II' conformation as opposed to the more common types I or II) and the other is a local electrostatic effect (responsible for distinguishing between the type I' and II' conformations). In the rare cases where a two-residue turn is found in a type I conformation, there exists a stabilizing feature (turn-protein interaction, a side-chain in a conformation that stabilizes type I, etc.) which compensates for the unfavorable twist of the turn relative to the beta-sheet.

Amino Acids↗