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G A Petsko

Publications and source records attributed to G A Petsko.

At least 55 records · Page 3Linked to original sources
PubMed1998

Crystallization and preliminary X-ray diffraction analysis of aspartate aminotransferase from Saccharomyces cerevisiae.

Diffraction-quality crystals of S. cerevisiae cytoplasmic aspartate aminotransferase have been obtained by the hanging-drop vapor-diffusion method in the presence of pyridoxal phosphate and maleic acid, sodium acetate, ammonium acetate and polyethylene glycol. The crystals have the symmetry of the orthorhombic space groups P212121 or P21212 with unit-cell dimensions a = 130.2, b = 134.6 and c = 98.7 A. Square rod-shaped crystals with dimensions of approximately 0.2 x 0.2 x 0.5 mm diffract to spacings of 2 A. The calculated value of the Matthews coefficient, Vm = 2.4 A3 Da-1, is consistent with four subunits of aspartate aminotransferase per asymmetric unit.

Aspartate Aminotransferases↗
PubMed1998

Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins.

3-Isopropylmalate dehydrogenase (IPMDH, E.C. 1.1.1.85) from the thermophilic bacterium Thermus thermophilus HB8 is homologous to IPMDH from the mesophilic Escherichia coli, but has an approximately 17 degreesC higher melting temperature. Its temperature optimum is 22-25 degreesC higher than that of the E. coli enzyme; however, it is hardly active at room temperature. The increased conformational rigidity required to stabilize the thermophilic enzyme against heat denaturation might explain its different temperature-activity profile. Hydrogen/deuterium exchange studies were performed on this thermophilic-mesophilic enzyme pair to compare their conformational flexibilities. It was found that Th. thermophilus IPMDH is significantly more rigid at room temperature than E. coli IPMDH, whereas the enzymes have nearly identical flexibilities under their respective optimal working conditions, suggesting that evolutionary adaptation tends to maintain a "corresponding state" regarding conformational flexibility. These observations confirm that conformational fluctuations necessary for catalytic function are restricted at room temperature in the thermophilic enzyme, suggesting a close relationship between conformational flexibility and enzyme function.

3-Isopropylmalate Dehydrogenase↗
PubMed1998

Inactivation and destruction of conserved Trp159 of Fe-superoxide dismutase from Porphyromonas gingivalis by hydrogen peroxide.

The superoxide dismutase (SOD) of Porphyromonas gingivalis, an obligate anaerobe, was purified from Escherichia coli (sodA sodB mutant) harboring the P. gingivalis SOD-encoding gene. The purified protein contained both iron and a small amount of manganese. Iron- and manganese-reconstituted SOD, which contained one of these metals exclusively, showed specific activities of 1000 and 1200 U/mg/mol of metals/subunit, respectively. These values were similar to the specific activity of the native enzyme purified from the recombinant E. coli strain. The Fe-reconstituted enzyme was inactivated by 10 mM hydrogen peroxide to about 5% of its original activity after a 15 min incubation at 25 degrees C at pH 7.8, whereas the Mn-reconstituted enzyme showed no inactivation after 80 min. A concomitant increase in absorbance at 320 nm was observed with inactivation of the Fe-reconstituted enzyme. Amino acid analysis of the inactivated Fe-reconstituted enzyme showed a decrease of about 0.7 residues of tryptophan/subunit, a value similar to the iron content of the iron-reconstituted enzyme. Three major peptides of the digests of the purified SOD with lysylendopeptidase were separated by a reverse-phase HPLC monitoring at 280 nm. One of the peptides, corresponding to the residues from Gly149 to Lys176, decreased in the HPLC eluent of the H2O2-inactivated SOD to 20% of the amount measured for native SOD. Since this peptide contains only one tryptophan residue, it was concluded that the decomposed tryptophan residue is Trp159, which is located midway between the third and fourth metal ligands, Asp157 and His161, and is conserved in aligned amino acid sequences of all known Fe-SODs and Mn-SODs. Based on these results, we propose that the differences in hydrogen peroxide sensitivities observed for the Fe-SODs and Mn-SODs may be caused by the difference in the identity of the active site metal in the Fe-SODs and Mn-SODs and a tuning of the properties of the iron center in the Fe-SODs.

Amino Acid Sequence↗
PubMed1998

Crystal structure of Saccharomyces cerevisiae cytosolic aspartate aminotransferase.

The crystal structure of Saccharomyces cerevisiae cytoplasmic aspartate aminotransferase (EC 2.6.1.1) has been determined to 2.05 A resolution in the presence of the cofactor pyridoxal-5'-phosphate and the competitive inhibitor maleate. The structure was solved by the method of molecular replacement. The final value of the crystallographic R-factor after refinement was 23.1% with good geometry of the final model. The yeast cytoplasmic enzyme is a homodimer with two identical active sites containing residues from each subunit. It is found in the "closed" conformation with a bound maleate inhibitor in each active site. It shares the same three-dimensional fold and active site residues as the aspartate aminotransferases from Escherichia coli, chicken cytoplasm, and chicken mitochondria, although it shares less than 50% sequence identity with any of them. The availability of four similar enzyme structures from distant regions of the evolutionary tree provides a measure of tolerated changes that can arise during millions of years of evolution.

Animals↗
PubMed1998

Crystal structures of L201A mutant of D-amino acid aminotransferase at 2.0 A resolution: implication of the structural role of Leu201 in transamination.

The leucine-to-alanine mutation at residue 201 of D-amino acid aminotransferase provides a unique enzyme which gradually loses its activity while catalyzing the normal transamination; the co-enzyme form is converted from pyridoxal 5'-phosphate to pyridoxamine 5'-phosphate upon the inactivation [Kishimoto,K., Yoshimura,T., Esaki,N., Sugio,S., Manning,J.M. and Soda,K. (1995) J. Biochem., 117, 691-696]. Crystal structures of both co-enzyme forms of the mutant enzyme have been determined at 2.0 A resolution: they are virtually identical, and are quite similar to that of the wild-type enzyme. Significant differences in both forms of the mutant are localized only on the bound co-enzyme, the side chains of Lys145 and Tyr31, and a water molecule sitting on the putative substrate binding site. Detailed comparisons of the structures of the mutant, together with that of the pyridoxamine-5'-phosphate form of the wild-type enzyme, imply that Leu201 would play a crucial role in the transamination reaction by keeping the pyridoxyl ring in the proper location without disturbing its oscillating motion, although the residue seems to not be especially important for the structural integrity of the enzyme.

Alanine↗
PubMed1997

The alrestatin double-decker: binding of two inhibitor molecules to human aldose reductase reveals a new specificity determinant.

It is generally expected that only one inhibitor molecule will bind to an enzyme active site. In fact, specific drug design theories depend upon this assumption. Here, we report the binding of two molecules of an inhibitor to the same active site which we observed in the 1.8 A resolution structure of the drug Alrestatin bound to a mutant of human aldose reductase. The two molecules of Alrestatin bind to the active site in a stacked arrangement (a double-decker). This stack positions the carboxylic acid of one drug molecule near the NADP+ cofactor at a previously determined anion binding site and the carboxylic acid of the second drug molecule near the carboxy-terminal tail of the enzyme. We propose that interactions of inhibitors with the carboxy-terminal loop of aldose reductase are critical for the development of inhibitors that are able to discriminate between aldose reductase and other members of the aldo-keto reductase superfamily. This finding suggests a new direction for the introduction of specificity to aldose reductase-targeted drugs.

Aldehyde Reductase↗
PubMed1997

Crystal structures of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus.

The basis of protein stability has been investigated by the structural comparison of themophilic enzymes with their mesophilic counterparts. A number of characteristics have been found that can contribute to the stabilization of thermophilic proteins, but no one is uniquely capable of imparting thermostability. The crystal structure of 3-isopropylmalate dehydrogenase (IPMDH) from the mesophiles Escherichia coli and Salmonella typhimurium have been determined by the method of molecular replacement using the known structure of the homologous Thermus thermophilus enzyme. The structure of the E. coli enzyme was refined at a resolution of 2.1 A to an R-factor of 17.3%, that of the S. typhimurium enzyme at 1.7 A resolution to an R-factor of 19.8%. The three structures were compared to elucidate the basis of the higher thermostability of the T. thermophilus enzyme. A mutant that created a cavity in the hydrophobic core of the thermophilic enzyme was designed to investigate the importance of packing density for thermostability. The structure of this mutant was analyzed. The main stabilizing features in the thermophilic enzyme are an increased number of salt bridges, additional hydrogen bonds, a proportionately larger and more hydrophobic subunit interface, shortened N and C termini and a larger number of proline residues. The mutation in the hydrophobic core of T. thermophilus IPMDH resulted in a cavity of 32 A3, but no significant effect on the activity and thermostability of the mutant was observed.

3-Isopropylmalate Dehydrogenase↗
PubMed1997

Determination of the structure of alanine racemase from Bacillus stearothermophilus at 1.9-A resolution.

The molecular structure of alanine racemase from Bacillus stearothermophilus was determined by X-ray crystallography to a resolution of 1.9 A. The alanine racemase monomer is composed of two domains, an eight-stranded alpha/beta barrel at the N-terminus, which includes residues 1-240, and a C-terminal domain essentially composed of beta-strand (residues 241-388). In the structure of the dimer the mouth of the alpha/beta barrel of one monomer faces the second domain of the other monomer. The pyridoxal 5'-phosphate (PLP) cofactor lies in and above the mouth of the alpha/beta barrel and is covalently linked via an aldimine linkage to Lys39, which is at the C-terminus of the first beta-strand of the alpha/beta barrel. This is the first example of a PLP cofactor binding in the active site of a alpha/beta barrel. A number of other residues are involved in maintaining the position of the PLP in the protein. Of these, Arg219 is the most interesting, as it forms a hydrogen bond with the pyridine nitrogen of the cofactor. This is the first known occurrence of such an interaction with PLP and is expected to influence the electron delocalization in the PLP-alanine intermediates. A second arginine residue, Arg136, donates a hydrogen bond to the phenolic oxygen of PLP and may be involved in the binding of substrate as well as stabilization of intermediates. Finally, Tyr265', from the second monomer, is postulated to be 2 proton donor to the carbanion intermediate.

Alanine Racemase↗
PubMed1997

Purification, catalytic properties and thermostability of 3-isopropylmalate dehydrogenase from Escherichia coli.

3-isopropylmalate dehydrogenase (IPMDH) from Escherichia coli was overexpressed, purified and crystallized. The enzyme was characterized and compared to its thermophilic counterpart from Thermus thermophilus strain HB8. As in the thermophile enzyme, the activity of E. coli IPMDH was dependent on the divalent cations, Mg2+ or Mn2+, with Mn2+ being the preferred cation. Activity was also strongly influenced by KCl: 0.3 M were necessary for the optimal activity. At 40 degrees C the K(m) of E. coli IPMDH was 105 microM for IPM and 321 microM for NAD, the kcat was 69 s-1. The half denaturation temperature was 64 degrees C, which was 20 degrees C lower than that of the thermophile enzyme.

3-Isopropylmalate Dehydrogenase↗
PubMed1997

A comparison between molecular dynamics and X-ray results for dissociated CO in myoglobin.

The distribution of carbon monoxide after photodissociation in the myoglobin haem pocket has been investigated using molecular dynamics simulations at 300 K. The results show that both intermediates (one close to the haem iron and one further away) observed in recent low temperature X-ray studies of photodissociated CO have a high probability of occurrence, even at ambient temperatures. The fact that the O of CO is oriented toward the haem iron in the closer intermediate provides an explanation for the slow rate of CO geminate rebinding. A refinement against X-ray data generated from the molecular dynamics simulations indicates that the CO has a broader distribution in the haem pocket than is apparent from the experimental electron density. This effect is likely to be general for systems containing highly mobile groups.

Algorithms↗
PubMed1997

Crystal structures of HINT demonstrate that histidine triad proteins are GalT-related nucleotide-binding proteins.

Histidine triad nucleotide-binding protein (HINT), a dimeric purine nucleotide-binding protein from rabbit heart, is a member of the HIT (histidine triad) superfamily which includes HINT homologues and FHIT (HIT protein encoded at the chromosome 3 fragile site) homologues. Crystal structures of HINT-nucleotide complexes demonstrate that the most conserved residues in the superfamily mediate nucleotide binding and that the HIT motif forms part of the phosphate binding loop. Galactose-1-phosphate uridylyltransferase, whose deficiency causes galactosemia, contains tandem HINT domains with the same fold and mode of nucleotide binding as HINT despite having no overall sequence similarity. Features of FHIT, a diadenosine polyphosphate hydrolase and candidate tumour suppressor, are predicted from HINT-nucleotide structures.

Amino Acid Sequence↗
PubMed1997

Sequence and homology model of 3-isopropylmalate dehydrogenase from the psychrotrophic bacterium Vibrio sp. I5 suggest reasons for thermal instability.

The leuB gene from the psychrotrophic strain Vibrio sp. I5 has been cloned and sequenced. The gene codes for 3-isopropylmalate dehydrogenase, a 360-residue, dimeric enzyme involved in the biosynthesis of leucine. Three recently solved homologous isopropylmalate dehydrogenase (IPMDH) crystal structures from thermophilic and mesophilic organisms have been used to build a homology model for the psychrotrophic IPMDH and to deduce the possible structural reasons for its decreased thermostability. According to our model the psychrotrophic IPMDH contains fewer stabilizing interactions than its mesophilic and thermophilic counterparts. Elements that have been identified as destabilizing in the comparison of the psychrotrophic, mesophilic and thermophilic IPMDHs are a smaller number of salt-bridges, a reduction in aromatic-aromatic interactions, fewer proline residues and longer surface loops. In addition, there are a number of substitutions of otherwise strictly conserved residues that can be linked to thermostability.

3-Isopropylmalate Dehydrogenase↗
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

For medicinal purposes.

Developments in techniques of pharmaceutical chemistry are poised to revolutionize the design of drugs beyond previous expectations. But what are our current expectations and what does the future hold?

Chemistry, Pharmaceutical↗
PubMed1996

Mechanism of the reaction catalyzed by mandelate racemase: structure and mechanistic properties of the D270N 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, with Lys 166 participating as the (S)-specific acid/base catalyst and His 297 participating as the (R)-specific acid/base catalyst. The dependence of kcat on pH for the racemization of both (R)- and (S)-mandelates suggests that the pKaS of the conjugate acids of Lys 166 and His 297 are both approximately 6.4 [Landro, J.A., Kallarakal, A.T., Ransom, S.C., Gerlt, J.A., Kozarich, J.W., Neidhart, D.J., Kenyon, G.L. (1991) Biochemistry 30, 9274-9281; Kallarakal, A.T., Mitra, B., Kozarich, J.W., Gerlt, J.A., Clifton, J.R., Petsko, G.A., & Kenyon, G.L. (1995) Biochemistry 34, 2788-2797]. Both acid/base catalysts are in close proximity to and approximately equidistant to the epsilon-ammonium group of Lys 164 and the essential Mg2+. The positive electrostatic potential provided by these cationic groups might be expected to increase the acidities of the cationic conjugate acids of the acid/base catalysts, thereby explaining the depressed pKa of Lys 166 but not the "normal" pKa of His 297. Asp 270 is hydrogen bonded of N delta of His 297 and, therefore, may allow the pKa of His 297 to be normal. In this paper we report the structural and mechanistic properties of the mutant in which Asp 270 is replaced with asparagine (D270N). The structure of D270N with (S)-atrolactate bound in the active site reveals no geometric alterations in the active site when compared to the structure of wild-type MR complexed with (S)-atrolactate, with the exception that the side chain of His 297 is tilted and displaced approximately 0.5 A away from Asn 270 and toward the (S)-atrolactate. The kcatS for both (R)- and (S)-mandelates are reduced approximately 10(4)-fold. In accord with the proposal that Asp 270 influences the pKa of His 297, in the (R)- to (S)-direction no ascending limb is detected in the dependence of kcat of pH; instead, kcat decreases from a low pH plateau as described by a pKa of 10. In the (S)- to (R)-direction the dependence of kcat of pH is a bell-shaped curve that is described by pKaS of 6.4 and 10. In analogy to the previously reported properties of the H297N mutant [Landro, J.A., Kallarakal, A.T., Ransom, S.C., Gerlt, J.A., Kozarich, J.W., Neidhart, D.J., & Kenyon, G.L. (1991) Biochemistry 30, 9274-9281], D270N catalyzes both the facile exchange of the alpha-proton of (S)- but not (R)-mandelate with solvent and the stereospecific elimination of bromide ion from (S)-p-(bromomethyl)mandalate. These observations suggest that His 297 and Asp 270 function as a catalytic dyad, with Asp 270 being at least partially responsible for the normal pKa of His 297 in wild-type MR.

Base Sequence↗