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Biomedical subjects

W N Lipscomb

Publications and source records attributed to W N Lipscomb.

At least 127 records · Page 7Linked to original sources
PubMed1978

Conformation of the mushroom toxin beta-amanitin in the crystalline state.

A single crystal X-ray diffraction analysis of beta-amanitin, a bicyclic octapeptide toxin isolated from the poisonous mushroom Amanita phalloides, shows that the molecule has distinct regions of hydrophilic and hydrophobic residues and two 18-membered rings. The study confirms the proposed chemical sequence and the configuration of the residues. All eight peptide groups are in the trans conformation. Four intramolecular hydrogen bonds, two strong and two weak, occur in the structure. The toxin cocrystallizes with seven water and three ethanol molecules and participates in an extensive hydrogen-bonding network. The crystal structure was solved by direct methods. The space group is P2(1)2(1)2(1), and unit cell dimensions are a = 14.004(3), b = 14.943(3), and c = 30.794(7) A.

Amanitins↗
PubMed1978

The effect of pH on the cooperative behavior of aspartate transcarbamylase from Escherichia coli.

Saturation curves of activity versus concentration were determined for aspartate transcarbamylase from Escherichia coli (EC 2.1.3.2) for the substrate L-aspartate at saturating carbamyl phosphate (4.8 mM) in buffered solution at pH values from 6.0 to 12.0. Hill coefficients were obtained from the sigmoidal curves. At pH values from 7.8 to 9.1, where substrate inhibition causes difficulties in the Hill approximation, our kinetic scheme includes substrate inhibition and residual activity in the abortive enzyme-substrate complex. The plot of Hill coefficient versus pH has pKalpha values of 7.4 and 9.8 at the half-maximum positions of the curve which has a plateau from pH 8.1 to 9.1. These pKalpha values may be associated with functional groups involved in the allosteric transition which activates the enzyme. A plot of [S]0.5 versus pH shows a pKalpha of 8.5, which may belong to a residue either at or near the aspartate binding site. At 50 mM aspartate concentration the pH-rate profile shows maxima at pH values of 8.8 and 10.0 (cf. Weitzman, P.D.J., and Wilson, I.B.(1966)J. Biol. Chem. 2418 5481-5488, who used 100 mM aspartate). However, when the pH-dependent substrate inhibition is included, the calculated Vmax--H curve is bell-shaped like that of the isolated catalytic subunit.

Aspartate Carbamoyltransferase↗
PubMed1978

Three-dimensional structures of aspartate carbamoyltransferase from Escherichia coli and of its complex with cytidine triphosphate.

X-ray diffraction studies to nominal resolutions of 3.0 A for unliganded aspartate carbamolytransferase (EC 2.1.3.2)(R32 crystal symmetry) and of 2.8 A for the complex of aspartate carbamoyltransferase with cytidine triphosphate (P321 crystal symmetry) have yielded traces of the polypeptide chains of the catalytic (C) and regulatory (R) chains in the hexameric C6R6 molecules. The independent molecular structures of the liganded and unliganded forms of the enzyme are very nearly identical. In the regulatory chain there is a CTP-binding domain that interacts with an adjacent regulatory subunit and a zinc-binding domain that interacts with the catalytic subunit. In the catalytic chain a polar domain shows interactions between adjacent pairs of C chains to form each trimer C3 while an equatorial domain shows intramolecular C3--C3 interactions. The active site is at or near the interface between adjacent C chains within the trimers. Probably each active center involves amino acid residues from adjacent C chains.

Aspartate Carbamoyltransferase↗
PubMed1978

Elimination of cooperativity in aspartate transcarbamylase by nitration of a single tyrosine residue.

In a previous report [Landfear, S. M., Lipscomb, W. N. & Evans, D.R. (1978) J. Biol. Chem. 253, 3988--3996] we demonstrated that tetranitromethane can be employed to nitrate a limited number of tyrosine residues in aspartate transcarbamylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2); such modification eliminates cooperativity, feedback inhibition, and enzymatic activity, and reduces binding of the feedback inhibitor cytidine triphosphate. Cooperativity is lost more rapidly than other properties, and this loss correlates with the nitration of a single tyrosine residue. In this paper, we describe the saturation kinetics of hybrid species constructed from nitrated subunits of one type (either catalytic or regulatory) and native subunits of the other type. We conclude that the modification responsible for loss of cooperativity is on the catalytic subunit. The tryptic peptide containing this modification has been isolated and identified.

Allosteric Regulation↗
PubMed1977

Functionally important arginine residues of aspartate transcarbamylase.

The reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity (Kantrowitz, E. R., and Lipscomb, W. N. (1976) J. Biol. Chem. 251, 2688-2695). If N-(phosphonacetyl)-L-aspartate is used to protect the active site, we find that phenylglyoxal causes destruction of the enzyme's susceptibility to activation by ATP and inhibition by CTP. Furthermore, CTP only minimally protects the regulatory site from reaction with this reagent. The modified enzyme still binds CTP although with reduced affinity. After reaction with phenylglyoxal, the native enzyme shows reduced cooperativity. The hybrid with modified regulatory subunits and native catalytic subunits exhibits slight heterotropic or homotropic properties, while the reverse hybrid, with modified catalytic subunits and native regulatory subunits, shows much reduced homotropic properties but practically normal heterotropic interactions. The decrease in the ability of CTP to inhibit the enzyme correlates with the loss of 2 arginine residues/regulatory chain (Mr = 17,000). Under these reaction conditions, 1 arginine residue is also modified on each catalytic chain (Mr = 33,000). Reaction rate studies of p-hydroxymercuribenzoate, with the liganded and unliganded modified enzyme suggest that the reaction with phenylglyoxal locks the enzyme into the liganded conformation. The conformational state of the regulatory subunit is implicated as having a critical role in the expression of the enzyme's heterotropic and homotropic properties.

Aldehydes↗
PubMed1977

Intramolecular interactions, enzyme activity and models.

The specificity of protein binding and the specificity-catalysis relationship in enzymes are analysed. Many enzymes use extended binding sites to achieve specificity and to create special environments which activate chemical groups on both the substrate and the enzyme itself. Aside from this effect, which is not adequately available in model compounds, the use of models is exemplified for several possibly separable effects in enzyme-substrate reactions. These include proximity (entropy loss on binding), locking into a productive binding mode, desolvation, electrostatic effects, changes of pKa by local environments, geometric strain, acid-base catalysis, and formation of other intermediates.

Binding Sites↗
PubMed1977

Interaction of tetraiodofluorescein with a modified form of aspartate transcarbamylase.

Low concentrations of the dye tetraiodofluorescein activate native aspartate transcarbamylase (aspartate carbomoyltransferase, carbomoylphosphate:L-aspartate carbomoyltransferase, EC 2.1.3.2), while high concentrations inhibit the enzyme's activity [Jacobsberg, L. B., Kantrowitz, E. R. & Lipscomb, W. N. (1975) J. Biol. Chem. 250, 9238-9249]. This dye is now shown to produce similar effects upon a modified form of aspartate transcarbamylase produced by Escherichia coli grown in a culture medium supplemented with thiouracil. Significantly, the ATP-induced activation is reduced in the modified form of the enzyme to the same extent as is the tetraiodofluorescein-induced activation. Thus, a relationship is demonstrated between the internal mechanisms by which ATP and tetraiodofluorescein activate aspartate transcarbamylase.

Adenosine Triphosphate↗
PubMed1976

Electronic structure and bonding of the amino acids containing first row atoms.

The electronic structures of the amino acids containing first row atoms have been determined for the zwitterionic form using an approximate self-consistent field method, partial retention of diatomic differential overlap. Various energetic quantities including certain proton affinities are present as are eigenvalues for the highest occupied and lowest unoccupied molecular orbitals. It is found that our method, in common with all methods employing minimum basis sets, yields eigenvalues for the highest occupied molecular orbital that are too high. The method does predict the location of this orbital correctly when compared to calculations employing larger basis sets. It is predicted that electron loss due to ionizing radiation should occur from the carboxylate group for the nonaromatic amino acids, while for tyrosine and tryptophan, electron loss should occur from the ring system. No choice between these two sites can be made for phenylalanine. Charge distributions have been obtained which show that only partial zwitterionic character is found in the backbone and that little delocalization of charge from the backbone to the side chain occurs. Localized molecular orbitals have been obtained using the Boys criteria and the bonding in the amino acids is disscussed in terms of these orbitals. Hybridization of various bonds and bond polarities are discussed as is the phenomenon of fractional bonding to carbon.

Amino Acids↗
PubMed1976

An essential residue at the active site of aspartate transcarbamylase.

Reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity. This modification reaction is markedly influenced by pH and is partially reversible upon dialysis. Carbamyl phosphate or carbamyl phosphate with succinate partially protect the catalytic subunit and the native enzyme from inactivation by phenylglyoxal. In the native enzyme complete protection from inactivation is afforded by N-(phosphonacetyl)-L-aspartate. The decrease in enzymatic activity correlates with the modification of 6 arginine residues on each aspartate transcarbamylase molecule, i.e. 1 arginine per catalytic site. The data suggest that the essential arginine is involved in the binding of carbamyl phosphate to the enzyme. Reaction of the single thiol on the catalytic chain with 2-chloromercuri-4-nitrophenol does not prevent subsequent reaction with phenylglyoxal. If N-(phosphonacetyl)-L-aspartate is used to protect the active site we find that phenylglyoxal also causes the loss of activation of ATP and inhibition by CTP. The rate of loss of heterotropic effects is exactly the same for both nucleotides indicating that the two opposite regulatory effects originate at the same location on the enzyme, or are transmitted by the same mechanism between the subunits, or both.

Amino Acids↗
PubMed1976

Molecular orbital studies of enzyme activity: catalytic mechanism of serine proteinases.

The catalytic activity of the serine proteinases is studied using molecular orbital methods on a model of the enzyme-substrate complex. A mechanism is employed in which Ser-195, upon donating a proton to the His-57-Asp-102 dyad, attacks the substrate to form the tetrahedral intermediate. As His-57 then donates a proton to the leaving group, the intermediate decomposes to the acyl enzyme. An analogous process takes place during deacylation, as a water molecule takes the place of Ser-195 as the nucleophile. The motility of the histidine is found to be an important factor in both steps. An attempt is made to include the effects of those atoms not explicitly included in the calculations and to compare the reaction rate of the proposed mechanism with that of the uncatalyzed hydrolysis. This mechanism is found to be in good agreement with structural and kinetic data.

Binding Sites↗
PubMed1975

Interaction of tetraiodofluorescein with aspartate transcarbamylase and its isolated catalytic and regulatory subunits.

The interaction of the dye tetraiodofluorescein with native aspartate transcarbamylase and its isolated subunits has been investigated by both binding and activity measurements at 4 and 23 degrees. At room temperature low concentrations of tetraiodofluorescein activate the native enzyme, but high concentrations inhibit the enzyme's activity. At the low temperature the native enzyme is inhibited by all concentrations of dye. Isolated catalytic subunit is very effectively inhibited at both temperatures. For the native enzyme these results are explained by 18 tetraiodofluorescein sites of approximately equal affinity (K = 7.3 X 10(-5) M) on each enzyme hexamer: one class of six sites at the nucleoside triphosphate effector binding sites is responsible for the activation, a second class which competes with the substrate carbamylphosphate causes the inhibition, and a third class does not interact with either the effectors or the substrates. Measurements of tetraiodofluorescein binding to isolated regulatory, catalytic, and p-hydroxymercuribenzoate-inactivated catalytic subunits support the above assignments. This scheme of tetraiodofluorescein binding sites successfully predicts the changes in the tetraiodofluorescein-aspartate transcarbamylase difference spectrum induced by the addition of various ligands. The activity changes induced by the dye are explained if the binding of a single tetraiodofluorescein molecule to one of the six regulatory sites activates all six of the catalytic sites, while while a dye molecule binding to any one of the catalytic sites inactivates only that catalytic site.

Aspartate Carbamoyltransferase↗
PubMed1975

Isolation and properties of a species produced by the partial dissociation of aspartate transcarbamylase from Escherichia coli.

A species produced by the reaction of aspartate transcarbamylase (C6R6) with 6 to 12 eq of p-hydroxymercuribenzoate was isolated by DEAE-Sephadex chromatography. Purified material was completely dissociated with mercurials and the relative amounts of catalytic (C) and regulatory (R) subunits were determined by three methods: (a) quantitative cellulose acetate electrophoresis; (b) Lowry analysis after separating the catalytic and regulatory subunits by sucrose gradient centrifugation; (c) dissociation of the species with sodium dodecyl sulfate and determination of the relative amounts of catalytic and regulatory chain by sodium dodecyl sulfate gel electrophoresis. All three methods gave consistent results, indicating that the molecule consists of 75% (by weight) catalytic chain and 25% regulatory chain. The molecular weight determined by gel filtration, sedimentation velocity, and sedimentation equilibrium experiments was found to be approximately 270,000. These observations establish that this species has the structure C6R4, and is produced by the release of a single regulatory dimer R2 from the intact aspartate transcarbamylase complex. This protein (C6R4) contains 20 cysteines and four zinc ions, consistent with the proposed subunit structure. The purified intermediate C6R4 contains no mercury. The parent molecule C6R6 can be reconstituted from C6R4 by incubation with isolated regulatory subunit (R2) in the presence of zinc and beta-mercaptoethanol. Titration of C6R4 yields an end point which corresponds to the addition of 1 mol of regulatory subunit (R2) per mol of C6R4. The intermediate is quite stable at neutral pH but tends to disproportionate into aspartate transcarbamylase and catalytic subunit after prolonged storage or at elevated pH. The kinetic properties of this species have been investigated. The specific activity of C6R4 is virtually identical with that of the native enzyme but the regulatory properties are substantially reduced. Both homotropic and heterotropic interactions are reduced but not abolished, indicating that the intact structure C6R6 is not required for the allosteric transitions involved in regulation.

Adenosine Triphosphate↗