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W N Lipscomb

Publications and source records attributed to W N Lipscomb.

At least 73 records · Page 4Linked to original sources
PubMed1990

Molecular structure of leucine aminopeptidase at 2.7-A resolution.

The three-dimensional structure of bovine lens leucine aminopeptidase (EC 3.4.11.1) complexed with bestatin, a slow-binding inhibitor, has been solved to 3.0-A resolution by the multiple isomorphous replacement method with phase combination and density modification. In addition, the structure of the isomorphous native enzyme has been refined at 2.7-A resolution, and the current crystallographic R factor is 0.169 for a model that includes the two zinc ions and all 487 amino acid residues comprising the asymmetric unit. The enzyme is physiologically active as a hexamer, which has 32 symmetry and is triangular in shape with a triangle edge length of 115 A and maximal thickness of 90 A. The monomers are crystallographically equivalent and each is folded into two unequal alpha/beta domains connected by an alpha-helix to give a comma-like shape with approximate maximal dimensions of 90 x 55 x 55 A3. The secondary structural composition is 40% alpha-helix and 19% beta-strand. The N-terminal domain (160 amino acids) mediates trimer-trimer interactions and does not appear to participate directly in catalysis. The C-terminal domain (327 amino acids) is responsible for catalysis and binds the two zinc ions, which are 2.88 A apart. The pair of metal ions is located near the edge of an eight-stranded, saddle-shaped beta-sheet. One zinc ion is coordinated by carboxylate oxygen atoms of Asp-255, Asp-332, and Glu-334 and the carbonyl oxygen of Asp-332. The other zinc ion is coordinated by the carboxylate oxygen atoms of Asp-255, Asp-273, and Glu-334. The active site also contains two positively charged residues, Lys-250 and Arg-336. The six active sites are themselves located in the interior of the hexamer, where they line a disk-shaped cavity of radius 15 A and thickness 10 A. Access to this cavity is provided by solvent channels that run along the twofold symmetry axes.

Amino Acid Sequence↗
PubMed1989

Structure of cyclo(-L-prolylglycyl-)2 trihydrate.

(C14H20N4O4)2.3H2O, Mr = 670.721, monoclinic, P2(1), a = 7.353 (2), b = 21.921 (7), c = 9.878 (2) A, beta = 96.77 (2) degrees, V = 1581.1 (1) A3, Z = 2, Dx = 1.409 g cm-3, lambda(Cu Kalpha) = 1.54178 A, mu = 8.22 cm-1, F(000) = 716, T = 293 K, R = 0.034 for 2456 unique observed reflections. The two independent copies of the tetrapeptide found in the asymmetric unit have similar structures, which are both consistent with the results of NMR studies of cyclo-(-L-Pro-Gly-)2 in solution. The structures are asymmetric and have a trans-cis-trans-cis peptide backbone, in which the two L-Pro-Gly peptide bonds are trans and the two Gly-L-Pro peptide bonds are cis. A detailed comparison with other cyclic tetrapeptides is given, and a brief comparison with the results of single-crystal X-ray structures of other cyclic oligopeptides containing L-proline alternating with glycine is presented.

Amino Acid Sequence↗
PubMed1989

Binding of sulfonamide and acetamide to the active-site Zn2+ in carbonic anhydrase: a theoretical study.

Self-consistent field molecular orbital (SCF MO) calculations at both 4-31G and STO-3G levels have been used to examine the binding conformations of sulfonamide and acetamide compounds to the active site of carbonic anhydrase. The results are as follows: (1) sulfonamide binds to the Zn2+ ion in its deprotonated form through the sulfonamide nitrogen to the fourth coordination site of the metal ion; (2) acetamide as neutral species binds to the basic form of the enzyme through the carbonyl oxygen to the fifth coordination site of the metal ion; and (3) the acetamidate ion binds to the acid form of the enzyme through the amide nitrogen to form a tetracoordinated metal complex with three histidine ligands. Analysis of the effects of individual active-site residues on the binding conformations of these inhibitors suggests that metal alone favors bidentate coordination of sulfonamidate and acetamidate complexes and that electron donation from three histidine ligands to the metal ion determines the formation of a tetracoordinated metal complex, which is further stabilized by the presence of Thr 199, as it receives one hydrogen bond from the sulfonamide NH- or from the acetamide NH- and donates a backbone NH hydrogen bond to a sulfonamide oxygen. The calculated binding conformation of sulfonamide and the hydrogen-bonding interactions between sulfonamide and the enzyme are consistent with the X-ray diffraction study of the AMSulf-HCA II complex. However, no X-ray structures are available for amide-HCA II complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetamides↗
PubMed1989

Binding of D-phenylalanine and D-tyrosine to carboxypeptidase A.

The structures of the complexes of carboxypeptidase A with the amino acids D-phenylalanine and D-tyrosine are reported as determined by x-ray crystallographic methods to a resolution of 2.0 A. In each individual study one molecule of amino acids binds to the enzyme in the COOH-terminal hydrophobic pocket: the carboxylate of the bound ligand salt links with Arg-145, and the alpha-amino group salt links with Glu-270. The carboxylate of Glu-270 must break its hydrogen bond with the native zinc-bound water molecule in order to exploit the latter interaction. This result is in accord with spectroscopic studies which indicate that the binding of D or L amino acids (or analogues thereof) allows for more facile displacement of the metal-bound water by anions (Bicknell, R., Schaffer, A., Bertini, I., Luchinat, C., Vallee, B. L., and Auld, D. S. (1988) Biochemistry 27, 1050-1057). Additionally, we observe a significant movement of the zinc-bound water molecule (approximately 1 A) upon the binding of D-ligands. We propose that this unanticipated movement also contributes to anion sensitivity. The structural results of the current x-ray study correct predictions made in an early model building study regarding the binding of D-phenylalanine (Lipscomb, W. N., Hartsuck, J. A., Reeke, G. N., Jr., Quiocho, F. A., Bethge, P. H., Ludwig, M. L., Steitz, T. A., Muirhead, H., and Coppola, J. C. (1968) Brookhaven Symp. Biol. 21, 24-90).

Binding Sites↗
PubMed1989

Structure of a single amino acid mutant of aspartate carbamoyltransferase at 2.5-A resolution: implications for the cooperative mechanism.

One of the many interactions important for stabilizing the T state of aspartate carbamoyltransferase occurs between residues Tyr240 and Asp271 within one catalytic chain. The functional importance of this polar interaction was documented by site-directed mutagenesis in which the tyrosine was replaced by a phenylalanine [Middleton, S. A., & Kantrowitz, E. R. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5866-5870]. In the Tyr240----Phe mutant, the aspartate concentration required to achieve half-maximum velocity is reduced to 4.7 from 11.9 mM for the native enzyme. Here, we report an X-ray crystallographic study of the Tyr240----Phe enzyme at 2.5-A resolution. While employing crystallization conditions identical with those used to grow cytidine triphosphate ligated T-state crystals of the native enzyme, we obtain crystals of the mutant enzyme that are isomorphous to those of the native enzyme. Refinement of the mutant structure to an R factor of 0.219 (only eight solvent molecules included) and subsequent comparison to the native T-state structure indicate that the quaternary, tertiary, and secondary structures of the mutant are similar to those for the native T-state enzyme. However, the conformation of Phe240 in one of the two crystallographically independent catalytic chains contained in the asymmetric unit is significantly different from the conformation of Tyr240 in the native T-state enzyme and similar to the conformation of Tyr240 as determined from the R-state structure [Ke, H.-M., Lipscomb, W. N., Cho, Y. J., & Honzatko, R. B. (1988) J. Mol. Biol. (in press)], thereby indicating that the mutant has made a conformational change toward the R state, localized at the site of the mutation in one of the catalytic chains.

Aspartate Carbamoyltransferase↗
PubMed1989

Crystal structure of the Glu-239----Gln mutant of aspartate carbamoyltransferase at 3.1-A resolution: an intermediate quaternary structure.

The structure of the unligated Glu 239----Gln mutant of Escherichia coli aspartate carbamoyltransferase (EC 2.1.3.2) has been determined to 3.1-A resolution and refined to a crystallographic residual of 0.22 in the space group P321. The unit-cell dimensions of the unligated enzyme are a = 122.3 A, c = 147.1 A. The c axis cell length is intermediate between the c axis lengths of the T (tense)(c = 142.2 A) and R (relaxed) (c = 156.2 A) state structures. Furthermore, the quaternary structure of the mutant enzyme is intermediate between the quaternary structures of the T form and the R form. The differences between the quaternary structures of the Glu-239----Gln and T-form enzymes can be described as follows: the separation between the catalytic trimers increases by approximately 1.5 A along the threefold axis, and they each rotate in opposite directions approximately 0.5 degree around the threefold axis, whereas the regulatory dimers rotate approximately 2 degrees around the twofold axes.

Aspartate Carbamoyltransferase↗
PubMed1989

Structural transitions in crystals of native aspartate carbamoyltransferase.

Screened precession x-ray photographs of crystals of native aspartate carbamoyltransferase (EC 2.1.3.2, from Escherichia coli) ligated with L-aspartate and phosphate reveal the presence of a crystal unit-cell dimension that is intermediate between the T (tense) and R (relaxed) states. Characterizing the intermediate (I) crystal is a c-axis unit-cell dimension of 149 A, halfway between the c-axis length of the T (c = 142 A) and R (c = 156 A) states, in the space group P321. Preservation of the P321 space group indicates that the intermediate crystal form retains a threefold axis of symmetry, and therefore the enzyme has at minimum a threefold axis; however, we do not know whether the molecular twofold axis is conserved. The I crystals are formed by soaking T-state crystals with L-aspartate and phosphate. By raising the concentration of L-aspartate we can further transform the I crystals, without fragmentation, to a form that has the same unit-cell dimensions as R-state crystals grown in the presence of N-(phosphonoacetyl)-L-aspartate.

Aspartate Carbamoyltransferase↗
PubMed1989

Molecular structure of fructose-1,6-bisphosphatase at 2.8-A resolution.

Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) from the cortex of pig kidney and its complexes with either fructose 2,6-bisphosphate (Fru-2,6-P2) or adenosine monophosphate (AMP) have been crystallized in the space group P3(2)21. The three-dimensional structure of the native enzyme has been solved at 3.0-A resolution by the multiple isomorphous replacement method and refined at 2.8-A resolution to a crystallographic R factor of 0.194. A total of 316 of 335 residues, omitting disordered regions 1-5 and 54-67, have been built into the monomer, which has average dimensions of about 30 A by 50 A by 35 A. Four monomeric units aggregate into a molecular tetramer with D2 symmetry, which approximates a disk about 35 A thick. Each monomer consists of about 33% alpha-helix, 23% beta-strand, and 6% beta-turn. Four sites for Fru-2,6-P2 and two major sites for AMP binding per tetramer have been identified by difference Fourier techniques. The binding site for Fru-2,6-P2 is shared by two neighboring monomers and consists of side-chain atoms of Asn-212, Tyr-244, Tyr-264, and Lys-274; backbone atoms of Gly-246 through Met-248; and only Arg-243 from the adjacent subunit. In addition, Asn-125, Tyr-215, and Lys-269 are located within a distance of about 5 A of Fru-2,6-P2. A negatively charged pocket near this binding site includes Asp-118, Asp-121, Glu-280, Glu-97, and Glu-98. The AMP binding site is located near Val-17, Gln-20, Gly-21, Ala-24 through Met-30, Lys-112, Tyr-113, Arg-140, and Met-177.

Animals↗
PubMed1988

Complex of N-phosphonacetyl-L-aspartate with aspartate carbamoyltransferase. X-ray refinement, analysis of conformational changes and catalytic and allosteric mechanisms.

The allosteric enzyme aspartate carbamoyltransferase of Escherichia coli consists of six regulatory chains (R) and six catalytic chains (C) in D3 symmetry. The less active T conformation, complexed to the allosteric inhibitor CTP has been refined to 2.6 A (R-factor of 0.155). We now report refinement of the more active R conformation, complexed to the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) to 2.4 A (R-factor of 0.165, root-mean-square deviations from ideal bond distances and angles of 0.013 A and 2.2 degrees, respectively). The antiparallel beta-sheet in the revised segment 8-65 of the regulatory chain of the T conformation is confirmed in the R conformation, as is also the interchange of alanine 1 with the side-chain of asparagine 2 in the catalytic chain. The crystallographic asymmetric unit containing one-third of the molecule (C2R2) includes 925 sites for water molecules, and seven side-chains in alternative conformations. The gross conformational changes of the T to R transition are confirmed, including the elongation of the molecule along its threefold axis by 12 A, the relative reorientation of the catalytic trimers C3 by 10 degrees, and the rotation of the regulatory dimers R2 about the molecular twofold axis by 15 degrees. No changes occur in secondary structure. Essentially rigid-body transformations account for the movement of the four domains of each catalytic-regulatory unit; these include the allosteric effector domain, the equatorial (aspartate) domain, and the combination of the polar (carbamyl phosphate) and zinc domain, which moves as a rigid unit. However, interfaces change, for example the interface between the zinc domain of the R chain and the equatorial domain of the C chain, is nearly absent in the T state, but becomes extensive in the R state of the enzyme; also one catalytic-regulatory interface (C1-R4) of the T state disappears in the more active R state of the enzyme. Segments 50-55, 77-86 and 231-246 of the catalytic chain and segments 51-55, 67-72 and 150-153 of the regulatory chain show conformational changes that go beyond the rigid-body movement of their corresponding domains. The localized conformational changes in the catalytic chain all derive from the interactions of the enzyme with the inhibitor PALA; these changes may be important for the catalytic mechanism. The conformation changes in segments 67-72 and 150-153 of the regulatory chain may be important for the allosteric control of substrate binding. On the basis of the conformational differences of the T and R states of the enzyme, we present a plausible scheme for catalysis that assumes the ordered binding of substrates and the ordered release o

Amino Acid Sequence↗
PubMed1988

Hydration of CO2 by carbonic anhydrase: intramolecular proton transfer between Zn2+-bound H2O and histidine 64 in human carbonic anhydrase II.

The energy barrier for the intramolecular proton transfer between zinc-bound water and His 64 in the active site of human carbonic anhydrase II (HCA II) has been studied at the partial retention of diatomic differential overlap (PRDDO) level. The most important stabilizing factor for the intramolecular proton transfer is the zinc ion, which lowers the pKa of zinc-bound water and electrostatically repels the proton. The energy barrier of 127.5 kcal/mol for proton transfer between a water dimer is completely removed in the presence of the zinc ion. The zinc ligands, which donate electrons to the zinc ion, raise the barrier slightly to 34 kcal/mol for a 4-coordinated zinc complex including three imidazole ligands from His 94, His 96, and His 119 and to 54 kcal/mol for the 5-coordinated zinc complex including the fifth water ligand. A few model calculations indicate that these energy barriers are expected to be reduced to within experimental range (approximately 10 kcal/mol) when large basis set, correlation energies, and molecular dynamics are considered. The proton-transfer group, which functions as proton receiver in the intramolecular proton transfer, helps to attract the proton; and the partially ordered active site water molecules are important for proton relay function.

Binding Sites↗
PubMed1988

Escherichia coli aspartate transcarbamylase: the relation between structure and function.

The x-ray structures of the allosteric enzyme aspartate transcarbamylase from Escherichia coli have been solved and refined for both allosteric forms. The T form was determined in the presence of the heterotropic inhibitor cytidine triphosphate, CTP, while the R form was determined in the presence of the bisubstrate analog N-phosphonacetyl-L-aspartate. These two x-ray structures provide the starting point for an understanding of how allosteric enzymes are able to control the rates of metabolic pathways. Insights into the mechanisms of both catalysis and homotropic cooperativity have been obtained by using site-directed mutagenesis to probe residues thought to be critical to the function of the enzyme based on these x-ray structures.

Allosteric Regulation↗
PubMed1988

Three-dimensional structure of carbamoyl phosphate and succinate bound to aspartate carbamoyltransferase.

The three-dimensional structure of the ternary complex of carbamoyl phosphate, succinate, and aspartate carbamoyltransferase (EC 2.1.3.2) has been determined to 2.6-A resolution. The binding of the phosphate of carbamoyl phosphate is similar to the binding of the phosphonate of N-(phosphonoacetyl)-L-aspartate (PALA); interacting with the carboxylates of succinate are some of the same residues that interact with the carboxylates of PALA. The amino group of carbamoyl phosphate donates hydrogen bonds to the main-chain carbonyls of residues Pro-266 and Leu-267 and the side-chain carbonyl of Gln-137. In comparing the structure of the active sites in the PALA-enzyme complex to the active sites in the carbamoyl phosphate-succinate-enzyme complex, we find that they are similar.

Aspartate Carbamoyltransferase↗
PubMed1987

Hydration of carbon dioxide by carbonic anhydrase: internal proton transfer of Zn2+-bound HCO3-.

Proton transfer within HCO3- has been examined under various conditions through molecular orbital methods: partial retention of diatomic differential overlap and 4-31G self-consistent field programs. These conditions include the absence or presence of Zn2+, Zn2+(NH3)3, or a water ligand on Zn2+. In addition, 4-31G+ and some MP2/4-31G results are obtained. The use of Be2+ to simulate Zn2+ reproduces reaction pathways and energy barriers, except for marginal cases. The barrier of 35.6 kcal/mol for direct internal proton transfer is reduced to 3.5 kcal/mol when one water molecule, not bound to Zn2+, is included for proton relay and to 1.4 kcal/mol when two such water molecules are included. In the enzyme, either Thr-199 or solvent molecules could perform this relay function. Our results favor this facilitated proton transfer over a mechanism in which Zn2+-bound OH- attacks CO2, a bidentate intermediate forms, and the OH moiety of the resulting HCO3- dissociates from Zn2+, thus leaving one of the oxygens of the original CO2 as a ligand to Zn2+.

Bicarbonates↗
PubMed1987

Structural asymmetry in the CTP-liganded form of aspartate carbamoyltransferase from Escherichia coli.

The protein and solvent structure of the CTP-liganded form of aspartate carbamoyltransferase from Escherichia coli yields an R-factor of 0.155 for data to a resolution of 2.6 A. The model has 7353 protein atoms, 945 sites for solvent, and two molecules of CTP. A total of 25 of the 912 residues of the model exist in more than one conformation. The root-mean-square deviation of bond lengths and angles from their ideal values is 0.013 A and 2.1 degrees, respectively. The model reported here reflects a correction in the trace of the regulatory chain. One molecule of CTP binds to each of the two regulatory chains of the asymmetric unit of the crystal. The interactions between the pyrimidine of each CTP molecule and the protein are similar. The 4-amino group of CTP binds to the carbonyl groups of residues 89 (tyrosine) and 12 (isoleucine) of the regulatory chain. The nitrogen of position 3 of the pyrimidine binds to the amide group of residue 12; the 2-keto group binds to lysine 60. The 2'-OH group of the ribose forms hydrogen bonds with lysine 60 and the carbonyl group of residue 9 (valine). The binding of the phosphate groups of CTP to the regulatory chain probably reflects an incomplete association of CTP with the enzyme at pH 5.8. A lattice contact influences the interaction between the triphosphate group of one CTP molecule and the protein. For the other CTP molecule, only lysine 94 binds to the phosphate groups of CTP. Of the two regulatory and two catalytic chains of the asymmetric unit of the crystal, there are only two significant violations of non-crystallographic symmetry. The active site in the vicinity of arginine 54 of one catalytic chain is larger than the active site of its non-crystallographic mate. The "expanded" cavity accommodates four solvent molecules in the vicinity of arginine 54 as opposed to two molecules of water for the "contracted" cavity. Furthermore, arginine 54 in the "expanded" pocket adopts two conformations, either hydrogen-bonding to glutamate 86 or to the phenolic oxygen atom of tyrosine 98; residues 86 and 98 are in a catalytic chain related by 3-fold symmetry to the catalytic chain of arginine 54. In the "contracted" pocket, arginine 54 binds only to glutamate 86.(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric Regulation↗
PubMed1987

The catalytic mechanism of Escherichia coli aspartate carbamoyltransferase: a molecular modelling study.

Based on molecular modelling study, we propose that the reaction between L-aspartate an carbamoylphosphate, catalyzed by E. coli aspartate carbamoyltransferase, may proceed via a tetrahedral intermediate and that the breakdown of the intermediate is facilitated by an intramolecular proton transfer between the amino group of L-aspartate and a terminal phosphate oxygen of carbamoylphosphate.

Aspartate Carbamoyltransferase↗
PubMed1987

2.5 A structure of aspartate carbamoyltransferase complexed with the bisubstrate analog N-(phosphonacetyl)-L-aspartate.

In an X-ray diffraction study using the method of multiple isomorphous replacement, the structure of aspartate carbamoyltransferase (EC 2.1.3.2) complexed with the bisubstrate analog N-(phosphonacetyl)-L-aspartate (PALA) has been solved to 2.5 A. Ten rounds of model building and 123 cycles of restrained reciprocal space refinement have resulted in a model containing 94.4% of the theoretical atoms of the protein-inhibitor complex with an R-factor of 0.231. The fit of the model to the density is excellent, except for occasional side-chains and two sections of the regulatory chains that may be disordered. The electron density for the PALA molecule is readily identifiable for both catalytic (c) chains of the asymmetric unit and bonding interactions with several important residues including Ser52, Arg54, Thr55, Ser80, Lys84, Arg105, His134, Arg165, Arg229 and Gln231 are apparent. The carboxylate groups of the PALA molecule are in a nearly cis conformation. Gross quaternary changes between the T and R forms are noted and in agreement with earlier work from this laboratory. Namely, in the new structure the catalytic trimers move apart by 12 A along the 3-fold axis of the enzyme and relocate by 10 degrees relative to each other, adopting a more eclipsed position. The regulatory (r) chains in the new structure reorient about their 2-fold axis by 15 degrees. Large tertiary changes that include domain migration and rearrangement are also present between these two forms. In the R form both domains of the catalytic chain relocate closer to each other in order to bind to the inhibitor. The polar domain seems to bind primarily to the carbamoyl phosphate moiety of PALA, and the equatorial domain binds primarily to the L-aspartate moiety. Other changes in tertiary structure bring the 80s loop (from an adjacent catalytic chain) and the 240s loop into a position to interact with the PALA molecule. Changes have been searched for in all interface regions of the enzyme. While the C1-C4 and C1-R4 regions have been completely altered, most of the other interchain interfaces are similar in the T and R forms. The intrachain interfaces, between domains of the same catalytic chains, have undergone some reorganization as these domains move closer to each other when the inhibitor is bound. This new structure allows a reinterpretation of genetic and chemical modification studies done to date.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1987

Mechanism of carboxypeptidase A: hydration of a ketonic substrate analogue.

The structure of the complex between carboxypeptidase A alpha (EC 3.4.17.1) and the ketonic substrate analogue 5-benzamido-2-benzyl-4-oxopentanoic acid (BOP) has been determined by x-ray crystallographic methods to a resolution of 1.7 A (final R = 0.191). Interestingly, BOP was observed to bind to the active site of carboxypeptidase A alpha as the covalent hydrate adduct. Because BOP is probably less than 0.2% hydrated in aqueous solution, this result was unexpected. One possibility is that the zinc-bound water of the native enzyme added to the ketone carbonyl. Alternatively, the enzyme may preferentially scavenge the hydrated ketone as it is continuously maintained at equilibrium in the solution in which the carboxypeptidase A alpha crystals were immersed. In either case, this mode of binding of BOP to carboxypeptidase A alpha provides an example of the preferred binding of a model of a structure along the reaction coordinate of a hydrolytic reaction.

Carboxypeptidases↗