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W N Lipscomb

Publications and source records attributed to W N Lipscomb.

At least 145 records · Page 8Linked to original sources
PubMed1975

Molecular orbital studies of enzyme activity: I: Charge relay system and tetrahedral intermediate in acylation of serine proteinases.

The charge relay ststem and its role in the acylation of serine proteinases is studied using the partial retention of diatomic differential overlap (PRDDO) technique to perform approximate ab initio molecular orbital calculations on a model of the enzyme-substrate complex. The aspartate in the charge relay system is seen to act as the ultimate proton acceptor during the charging of the serine nucleophile. A projection of the potential energy surface is obtained in a subspace corresponding to this charge transfer and to the coupled motions of active site residues and the substrate. These results together with extended basis set results for cruder models suggest that a concerted transfer of protons from Ser-195 to His-57 and from His-57 to Asp-102 occurs with an energy barrier of 20-25 kcal/mole (84-105 kJ/mole). The subsequent nucleophilic attack on the scissile peptide linkage by the charged serine is then seen to proceed energetically downhill to the tetrahedral intermediate. The formation of the tetrahedral intermediate from the Michaelis complex is calculated to be nearly thermoneutral.

Acylation↗
PubMed1974

Complex of aspartate carbamoyltransferase from Escherichia coli with its allosteric inhibitor, cytidine triphosphate: electron density at 5.9-angstroms resolution.

Following our earlier determination of the three-dimensional structure of aspartate carbamoyltransferase (EC 2.1.3.2; carbamoylphosphate: L-aspartate carbamoyltransferase) to 5.5-A resolution [S. G. Warren, B. F. P. Edwards, D. R. Evans, D. C. Wiley & W. N. Lipscomb (1973) Proc. Nat. Acad. Sci. USA 70, 1117-1121], we report here, from a different crystal form, the three-dimensional structure at 5.9 A of this enzyme complexed with its allosteric inhibitor, cytidine triphosphate. Location of the major binding site of this inhibitor within each of the six regulatory chains is made secure by comparison of these results with those obtained upon binding of 5-iodocytidine triphosphate to the enzyme. Conformational changes in the aspartate carbamoyltransferase molecule when this inhibitor binds are described briefly at 5.9-A resolution.

Allosteric Regulation↗
PubMed1974

An intermediate complex in the dissociation of aspartate transcarbamylase.

The multisubunit enzyme aspartate transcarbamylase consists of six copies of two types of polypeptide chains, catalytic (C) and regulatory (R). A complex formed by the partial dissociation of this enzyme has been isolated. This species, which has the structure C(6)R(4), is a likely intermediate in the stepwise dissociation of aspartate transcarbamylase induced by mercurials. The formation of the complex is the result of the release of a single regulatory dimer (R(2)) from the parent molecule.The specific activity of the intermediate is essentially the same as that of aspartate transcarbamylase. By contrast, both homotropic and heterotropic interactions are reduced, but not abolished. These observations suggest that the allosteric transitions involved in the control mechanisms do not require the intact structure C(6)R(6).

Allosteric Regulation↗
PubMed1974

Localized molecular orbital description of nitrogen lone pairs.

The Boys localization procedure is applied to several molecules possessing nitrogen lone pairs. The Boys structures show a tendency for the N lone pairs to participate in bonding to nearby electron sinks. For example, the Boys structure for planar formamide has two equivalent tau-bond orbitals between the carbonyl carbon and nitrogen atoms. The change in this description with variation of geometry at nitrogen is treated, and analogous Edmiston-Ruedenberg localizations on formamide are included for comparison.

Journal Article↗
PubMed1973

Aqueous central cavity in aspartate transcarbamylase from Escherichia coli.

A three-dimensional x-ray diffraction study of aspartate transcarbamylase to 5.5-angstrom resolution, with the aid of four isomorphous heavy atom derivatives, indicates the presence of a central aqueous cavity approximating an oblate spheroid about 25 by 50 by 50 angstroms in dimension, within a molecule about 90 by 110 by 110 angstroms in largest dimensions.

Aspartate Carbamoyltransferase↗
PubMed1973

Enzymatic activities of carobxypeptidase A's in solution and in crystals.

Activities of all known forms of bovine carboxypeptidase A's (alpha having 307, beta having 305, and gamma having 300 amino acids) are essentially the same in solution under given conditions. However, activities in the crystals differ. The A(alpha) crystals elongated along the a axis (a) have unit cell parameters a = 51.41 A, b = 59.89 A, c = 47.19 A, and beta = 97 degrees 35', (b) show about [unk] of the activity of the dissolved enzyme, and (c) have the same color of the arsanilazo Tyr 248 derivative in the crystalline and solution states, namely red at pH 8.2 and yellow at pH 7.4. The A(gamma) crystals elongated along the b axis (a) have unit cell parameters a = 50.9 A, b = 57.9 A, c = 45.0 A, and beta = 94 degrees 40', (b) show [unk] of the activity of the dissolved enzyme, and (c) show, in the arsanilazo Tyr 248 derivative, yellow crystals and red solution at pH 8.2. Although the detailed three-dimensional structure is known for the A(alpha) form described above, the structure of the A(gamma) form is as yet undertermined. A reasonable hypothesis is that the major part of the differences in crystal behaviors is due to differences in intermolecular (crystal-packing) interactions. In particular the movement of Tyr 248 may be somewhat hindered by these intermolecular contacts in the A(gamma) crystals, and in other crystalline forms which are elongated along the b axis. The movement observed in the x-ray diffraction studies, of the OH group of Tyr 248 by 12 A when the very slowly hydrolyzed substrate Gly-Tyr is bound to A(alpha) crystals, appears to be largely unhindered by intermolecular interactions. Examination of a three-dimensional space-filling structural model of the carboxypeptidase A molecule reveals that the phenolic oxygen of Tyr 248 can approach within 2 A of the Zn cofactor. This approach requires a movement by about 6 A of the polypeptide chain in the general region of Tyr 248. Moreover, the position of Tyr 248 when bonded to Zn can just be seen in the electron density map of the crystal structure at a level which, averaged over many unit cells, suggests some 15-25% of the enzyme is in this form at pH 7.4 and 4 degrees in the crystals of the x-ray diffraction study. It is probable that when the Zn-Tyr 248 bond is present the enzyme is catalytically inactive.

Amino Acids↗
PubMed1973

The reverse turn as a polypeptide conformation in globular proteins.

The reverse turn, involving four consecutive amino acids, as a tertiary conformation in globular proteins is defined in terms of dihedral angles, the C(1) (alpha)...C(4) (alpha) distance and the O(1)...H-N(4) hydrogen bond distance. In seven proteins we find 125 examples of turns, comprising 33% of the amino acids in these proteins, as compared with 34% of the residues forming helices and only 17% forming beta-sheets. The amino-acid compositions of turns, helices, and beta-sheets are analyzed in some detail. We find Asn and Gly mainly in turns, Pro in turns (and at the beginning of helices), and Glu in helices. In these turns a statistical survey indicates that 19% of Asp residues are in the first position, 33% of Pro residues are in the second position, 24% of Asn residues are in the third position, and 26% of Trp residues are in the fourth position.

Amino Acid Sequence↗
PubMed1973

Aspartate transcarbamoylase from Escherichia coli: electron density at 5.5 A resolution.

The allosteric enzyme, aspartate transcarbamoylase (EC 2.1.3.2), has previously been shown in our x-ray diffraction studies to have D(3)-32 symmetry. There are six catalytic (C) and six regulatory (R) chains in the molecular complex (R(6)C(6)). Our three-dimensional x-ray diffraction study of this enzyme (R32, a = 131 A, c = 200 A) at 5.5 A resolution shows a spatial arrangement of the two catalytic trimers C(3) above and below an equatorial belt of three regulatory dimers R(2). The molecule is about 110 x 110 x 90 A in largest dimensions, and is shown here to contain a large central aqueous cavity about 50 x 50 x 25 A in size. Location of the single sulfhydryl of each catalytic chain, and correlation of its reactivity with enzymatic activity in the molecule, suggests that the nearby active sites are most probably accessible from the central cavity, but probably not directly from the external solution. The most obvious access to the central cavity consists of six channels, each about 15 A in diameter, near the regulatory region. A component of the regulatory mechanism may be modulation of access of substrates through these channels.

Allosteric Regulation↗
PubMed1972

Similarities between the conformation of arsanilazotyrosine 248 of carboxypeptidase A in the crystalline state and in solution.

Modification of carboxypeptidase A(gamma) crystals (Anson) with diazotized arsanilic acid specifically labels tyrosine 248; at pH 8.2 the modified enzyme gives yellow crystals, but a red solution. It has been suggested that arsanilazotyrosine 248 forms a complex with the Zn cofactor accounting for the red color in solution, but that a complex is not formed in the crystal. However, the crystal structure of carboxypeptidase A(gamma) is unknown. We show here that crystals of carboxypeptidase A(alpha), whose crystal structure has been determined, are red both in solution and in the crystalline state (at pH 8.2) after modification with diazotized arsanilic acid. These new data are of importance in relating the structure in the crystalline state to the catalytic mechanisms, as based on the x-ray diffraction evidence. The activity of carboxypeptidase A in the crystal and in solution has a ratio of only 1/3 for the alpha form, in contrast to the ratio of 1/300 for the gamma form, with carbobenzoxyglycyl-L-phenylalanine as a substrate.A pH-jump experiment monitored by stopped-flow kinetics in a split-beam apparatus has revealed a single exponential rate when a solution of arsanilazotyrosine 248 carboxypeptidase A(alpha) at pH 6.7 (yellow) is increased to pH 8.5 (red). The rate constants obtained in this experiment are 6.1 sec(-1) at 3.0 mg/ml and 7.2 sec(-1) at 1.6 mg/ml concentration of enzyme.

Animals↗
PubMed1972

The thiol group in the catalytic chains of aspartate transcarbamoylase.

The allosteric enzyme aspartate transcarbamoylase (EC 2.1.3.2) was previously shown to consist of two functionally distinct types of polypeptide chains. X-ray diffraction and chemical studies showed that there are six copies of both catalytic (C) and regulatory (R) chains, and that the intact molecular complex (C(6)R(6)) has D(3) symmetry. Organomercurials react preferentially with the four thiol groups on each R chain, dissociating the molecular complex. We show that 2-chloromercuri-4-nitrophenol reacts specifically and rapidly with the single C-chain thiol, which is believed to be near the catalytic site. This reaction inactivates the enzyme in solution and does not dissociate the molecular complex. Spectrophotometric titration and mercury analysis indicates that six molecules of this mercurial are firmly bound to the enzyme (R(6)C(6)), and crystallographic studies establish that only six sites, related by D(3) symmetry, are modified. The known low reactivity of this C-chain thiol with other sulfhydryl reagents, the unusual structural requirements in the reaction with 2-chloromercuri-4-nitrophenol, and the spectral properties of the resulting derivative provide insight into the environment of this thiol. Probably, at least one positively charged group of the enzyme is nearby, and the environment of this thiol is at least partially hydrophobic.

Allosteric Regulation↗
PubMed1972

Approximations to self-consistent field molecular wavefunctions.

Unparameterized and parameterized versions are outlined of a new method for approximating self-consistent field wavefunctions from first principles at the minimum basis set level for complex molecules containing hydrogen and first-row atoms. The Hartree-Fock self-consistent field equations for closed-shell molecules are solved, retaining all one-electron integrals, and approximating the two-electron Coulomb integrals, hybrid integrals, and exchange integrals of the form (i(A)j(A)[unk]i(A)j(A)) and (i(A)j(B)[unk]i(A)j(B)) for centers A and B. A symmetrically orthogonalized basis set is used and rotational invariance is achieved by transformation to local axes that are unique for atoms in anisotropic environments. Parameterization based upon first-principle self-consistent field wavefunctions for a large number of molecules yields F-matrix elements to 0.007 atomic units (au), density matrix elements to 0.007 electrons, orbital populations and atomic charges to 0.01-0.02 electrons, orbital energies to 0.01 au, and total energies to 0.02 au (all standard deviations), in computational times only a few times larger than those required for complete neglect of differential overlap calculations.

Journal Article↗