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W N Lipscomb

Publications and source records attributed to W N Lipscomb.

At least 109 records · Page 6Linked to original sources
PubMed1982

Interactions of metal-nucleotide complexes with aspartate carbamoyltransferase in the crystalline state.

We report the results of crystallographic difference maps at 3.0-A resolution of complexes of metal-nucleoside triphosphates with aspartate carbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli. The complexes Gd3+-ATP, Al3+-ATP, and Gd3+-CTP bind to the allosteric effector domain of the enzyme in nearly the same orientation as the metal-free nucleotides. The result is consistent with kinetic observations of nearly identical allosteric efficacy of ATP and CTP and their complexes with cations. The effector Gd3+-GTP, however, binds in a distinctly different conformation and location than does 8-bromoguanosine 5'-triphosphate, reported in a separate investigation [Honzatko, R. B. & Lipscomb, W.N. (1982) J. Mol. Biol. 160, 265-286]. The difference in the binding modes of Gd3+-GTP and the bromo derivative suggests a possible mechanism for the relief of allosteric inhibition of GTP due to metal cations. We observe no binding of metal-nucleoside triphosphates in the region of the phosphate crevice of aspartate carbamoyltransferase, consistent with the reduced ability of metal nucleotides to compete with carbamoyl phosphate for the active site. However, a single Gd3+ ion binds in the region of the active site as evidenced by strong density. The binding of cations near the active site probably causes the inhibition of catalysis observed in kinetics experiments reported earlier [Honzatko, R.B., Lauritzen, A.M. & Lipscomb, W.N. (1981) Proc. Natl. Acad. Sci. USA 78, 898-902].

Adenosine Triphosphate↗
PubMed1982

Extension of the method of partial retention of diatomic differential overlap to second row atoms and transition metals.

The method of partial retention of diatomic differential overlap (PRDDO) has been extended to the elements through the first transition series. A minimum basis set of Slatertype orbitals is employed, with optional 3d functions on atoms in the second major row. The 3d shell for transition metal atoms is a fixed-contracted double-zeta combination of Slater orbitals. The method retains the basic n(3) dependency inherent in PRDDO. Limited one-center parameterization and empirical two-center exchange integrals are included. The overall accuracy of the approach is comparable to that of the PRDDO method as previously applied to molecules containing first-row atoms only.

Journal Article↗
PubMed1982

Zn(II)-induced cooperativity of Escherichia coli ornithine transcarbamoylase.

The steady-state reaction of ornithine transcarbamoylase (ornithine carbamoyltransferase, carbamoyl phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) purified from the argI gene product of Escherichia coli strain K-12 exhibits Michaelis-Menten kinetics over an extended range of concentration for both L-ornithine and carbamoyl phosphate. In the presence of Zn2+, however, the saturation curve of L-ornithine becomes sigmoidal, revealing positive cooperativity for this anabolic enzyme. The kinetic data give a limiting Hill coefficient of 2.7 for this substrate at 0.3 mM Zn2+. The allosteric effect of Zn2+ on the enzyme is not altered by the concentration of carbamoyl phosphate, and the saturation curve of carbamoyl phosphate remains hyperbolic in the presence of the metal ion. At fixed substrate concentrations, initial velocity data obtained at 0.-0.3 mM Zn2+ indicate cooperative binding of the metal ion to ornithine transcarbamoylase; a Hill coefficient of 1.7 +/- 0.1 is found that is independent of the level of L-ornithine. These results suggest competitive and exclusive binding to the enzyme between L-ornithine and Zn2+ with conformational changes induced in the subunits of the enzyme only by the metal ligand. Neither Co2+ nor Cu2+ exerts an effect on the kinetic behavior of the enzyme. This finding reveals not only specific allosteric control of ornithine transcarbamoylase by Zn2+ but also the possibility of an interlocking metabolic regulation between the urea cycle and the pathway for pyrimidine biosynthesis.

Escherichia coli↗
PubMed1981

Kinetics of aspartate transcarbamylase from Escherichia coli for the reverse direction of reaction.

The reverse reaction of aspartate transcarbamylase in which phosphate or arsenate is first coupled to carbamyl aspartate, followed by elimination of aspartate, has been studied under conditions in which one product, aspartate, is removed. Aspartate is converted to oxalacetate by glutamate-oxalacetate transaminase, and the resulting oxalacetate is converted to malate by the NADH, NAD+ oxidoreductase enzyme malate dehydrogenase. Phosphate and carbamyl aspartate saturation curves are nonsigmoidal. The transition state analogue, N-phosphonacetyl-L-aspartate, activates this reverse reaction substantially. Reverse kinetic parameters of the Haldane type are characteristic of the T-state and correlated with the parameters of the usual forward reaction of the T-state. Phosphate and carbamyl aspartate do not alter the thiol reactivity or sedimentation coefficient of the enzyme. These five results indicate that, under the conditions of these experiments, the reverse reaction does not cause the allosteric transition. In a new assay for the forward reaction we couple phosphate production with NADP reduction using phosphorylase a, phosphoglucomutase, and glucose-6-phosphate dehydrogenase.

Adenosine Triphosphate↗
PubMed1981

Metal cation influence on activity and regulation of aspartate carbamoyltransferase.

At saturating carbamoylphosphate and nonsaturating aspartate concentrations, Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Al3+, and Gd3+ inhibit aspartate carbamoyltransferase (carbamoylphosphate:L-asparate carbamoyltransferase, EC 2.1.3.2) from EScherichia coli. When nucleotide triphosphates are present, these inhibitory effects are displaced to higher concentrations of cation. At lower levels of cation and saturating carbamoylphosphate concentration, Mg2+, Mn2+, Al3+, and Gd3+ partially relieve allosteric inhibition by GTP but have little influence on activation by ATP and inhibition by CTP. At nonsaturating carbamoylphosphate concentrations, however, Mg2+, Mn2+, Al3+, and Gd3+ increase enzymatic activity to 170% over the level when GTP alone is present. In addition, Mg2+, Mn2+, and Al3+ show enhancement of ATP activation by 120-130% but only slight relief of CTP inhibition. We suggest that three modes of action by the metal can account for the observed kinetic behavior. (i) In the absence of nucleotide, metals inhibit catalytic activity either by a direct interaction with the enzyme or indirectly by complexing carbamoylphosphate. (ii) The metal-nucleotide complex interacts allosterically with the enzyme to enhance enzymatic activity relative to that produced by the free nucleotide, as noted above. (iii) By chelating to nucleotides, the metal diminishes their tendency to bind competitively at the carbamoylphosphate portion of the active site, as shown particularly by experiments on the catalytic subunit.

Aspartate Carbamoyltransferase↗
PubMed1981

Zinc environment and cis peptide bonds in carboxypeptidase A at 1.75-A resolution.

The structure of the metalloenzyme carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) has been refined at 1.75 A by a restrained least-squares procedure to a conventional crystallographic R factor of 0.162. Significant results of the refined structure relative to the catalytic mechanism are described. In the native enzyme, the zinc coordination number is five (two imidazole N delta 1 nitrogens, the two carboxylate oxygens of glutamate-72, and a water molecule). In the complex (at 2.0-A resolution) of carboxypeptidase A with the dipeptide glycyl-L-tyrosine, however, the water ligand is replaced by both the carbonyl oxygen and the amino nitrogen of the dipeptide. The amino nitrogen also statistically occupies a second position near glutamate-270. Consequently, the coordination number of zinc may vary from five to six in carboxypeptidase A-substrate complexes. Implications of these results for the catalytic mechanism of carboxypeptidase A are discussed. In addition, three cis peptide bonds, none of which involves proline as the amino nitrogen donor, have been located fairly near the active site.

Animals↗
PubMed1981

Binding of ligands to the active site of carboxypeptidase A.

We compare the detailed binding modes of the 39-amino acid inhibitor from potatoes, glycyl-L-tyrosine, the ester analogue CH3OC6H4(CO)CH2CH(CO2(-))C6H5, and indole acetate to the exopeptidase carboxypeptidase A (EC 3.4.17.1). In the potato inhibitor, cleavage of the COOH-terminal glycine-39 leaves a new carboxylate anion of valine-38 having one oxygen on zinc and the other as a receptor of a hydrogen bond from tyrosine-248 of carboxypeptidase. Tyrosine-248 also receives a hydrogen bond from the amide proton of the originally penultimate peptide bond between tyrosine-37 and valine-38. This hydrogen bond suggests product stabilization which is available to peptides and depsipeptides but not to esters lacking an equivalent peptide bond (nonspecific esters). Also, this structure may represent the intermediate binding step for the uncleaved substrate as it moves along the binding subsites. In particular, this may be the binding mode for the substrate after association of the COOH-terminal region of the substrate with the residues at binding subsite S2 (tyrosine-198, phenylalanine-279, and arginine-71) and preceding entry into the catalytic site S1'. These stabilized complexes allow some understanding of the effect of indole acetate, shown here to bind in the pocket at S1', as a competitive inhibitor for esters (for which entry into S1' precedes the rate-determining catalytic step for hydrolysis) and as a noncompetitive inhibitor for peptides (for which entry into S1' is rate limiting). These results, including the binding mode of the ester analogue, are consistent with the original proposal from x-ray studies that both esters and peptides are cleaved with the carboxy terminus at S1', although not necessarily by the same chemical steps.

Binding Sites↗
PubMed1981

A survey of x-ray diffraction studies of enzyme-other molecule interactions as possible models for receptor sites.

In the absence of a structure for a hormone-receptor complex, one may ask what systems of known structure are most likely to provide information about hormone interactions. Here, I discussed enzyme-substrate, enzyme-(protein) inhibitor, enzyme fragment (S peptide), and antibody-hapten (or antigen) interactions as possible models. Following a study of a fairly inflexible hormone (insulin) and of a flexible hormone (glucagon), I commented on probable binding regions. Finally, my conclusion was that, at present, allosteric enzymes have many of the characteristics thought to be present in those hormone-receptor interactions which activate enzymes. This model does not necessarily apply in detail to examples of hormone interactions that affect permeability of the cell wall or activate genetic processes.

Antibodies↗
PubMed1980

Structure of potato inhibitor complex of carboxypeptidase A at 5.5-A resolution.

The complex of the 39-amino inhibitor (potato) of bovine carboxypeptidase A (carboxypeptidase; peptidyl-L-amino-acid hydrolase, EC 3.4.12.2) was crystallized in space group P32. There are two protein-inhibitor complexes in the asymmetric unit. These crystals exhibited pseudo-P3221 symmetry due to twinning about the a3 axis. Heavy atom difference Patterson maps and rotation functions indicated, however, that the noncrystallographic twofold axis that relates these two complexes is nearly coincident with the a3 axis. Consequently, to a good approximation at low resolution, the space group of the complex is P3221 and the effects of twinning may be ignored. The structure was solved by using multiple isomorphous replacement and molecular replacement techniques. At 5.5-A resolution, the multiple isomorphous replacement map was readily interpretable in terms of the known native carboxypeptidase A structure plus extra density around the active site. The position of this extra density is consistent with the binding mode for extended substrate proposed from earlier model building studies with the native enzyme (Lipscomb, W.N., Hartsuck, J.A., Reeke, G.N., Quiocho, F.A. Bethge, P.H., Ludwig, M.L., Steitz, T.A., Muirhead, H. & Coppola, J.C. (1968) Brookhaven Symp. Biol. 21, 24-90).

Animals↗
PubMed1980

Structure of an actively exchanging complex between carboxypeptidase A and a substrate analogue.

An x-ray diffraction study at 2.8 A resolution has yielded the structure of a complex between bovine carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) and (-)-2-benzyl-3-p-methoxybenzoylpropionic acid. This substrate is an analogue of N-(p-methoxy)-benzoylphenylalanine, in which the amide NH is replaced by CN2. T. Sugimoto and E T. Kaiser (1979) J. Am. Chem. Soc. 101, 39469--3951] have shown that this complex catalyzes stereospecific exchange of that proton of the CH2 group which is in the R configuration. Our structure of this complex suports the model proposed by Sugimoto and Kaiser and is very similar to the productive peptide binding mode suggested by Lipscomb et al. [Lipscomb, W. N., Hartsuck, J. A., Reeke, G. N., Quiocho, F. A., Bethge, P. A., Ludwig, M. L., Steitz, T. A., Muirhead, H. & Coppola. J. C. (1968) Brookhaven Symp. Biol. 21, 24--90]. The proposed roles of glutamic acid 270 in the proton exchange and the interaction of zinc with the carbonyl group of the substrate are consistent with the observed structure.

Animals↗
PubMed1980

Carboxypeptidase A mechanisms.

The mode of binding of a ketonic substrate, which is an analogue of esters in which the O of the scissile bond is replaced by CH2, to carboxypeptidase A is similar to that of Gly-Tyr. The site is S'1, with the side chain in the pocket of the enzyme, the carboxylate salt-linked to Arg-145, and the carbonyl group bound to Zn. Thus, esters are probably cleaved at the peptide cleavage site, although not necessarily with the same rate-controlling step or by the same detailed mechanism. The large differences found between the behavior of the enzyme in solution and in one crystalline phase do not apply to a different crystalline phase.

Binding Sites↗
PubMed1980

Structure of the potato inhibitor complex of carboxypeptidase A at 2.5-A resolution.

The structure of the complex between the proteolytic enzyme carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) and the 39-amino-acid carboxypeptidase A inhibitor from potatoes has been determined at 2.5-A resolution. A combination of multiple isomorphous replacement, molecular replacement, and noncrystallographic symmetry averaging techniques was used to solve the structure. The chain trace of the inhibitor and details of the binding interactions in the complex are described. A surprising aspect of the complex is that the carboxy-terminal peptide bond of the inhibitor has been hydrolyzed, and the carboxy-terminal glycine is trapped in the binding pocket of carboxypeptidase A. Consequently, the complex resembles a stage in the catalytic mechanism after hydrolysis of the peptide bond. The ring of tyrosine-248, which is known to undergo large conformational changes upon substrate binding, is in the "down" position and interacts with the inhibitor in the complex.

Binding Sites↗
PubMed1979

Conformations and electronic structures of oxidized and reduced isoalloxazine.

The conformations of oxidized and reduced isoalloxazine have been examined by a molecular orbital method, PRDDO (partial retention of diatomic differential overlap). The angle theta of fold about the N...N line of the central ring is zero for the planar oxidized form, but a bend of theta = 10 degrees requires only 2 kcal/mol. On the other hand, the reduced form is nonplanar (theta approximately 15 degrees), and the barrier for reversal of this bend is 4 kcal/mol, comparable with that in simple amines. Molecular properties and reactivity are interpreted in terms of charge and orbital distributions, and localized molecular orbitals have been derived by the method of Boys.

Flavins↗
PubMed1979

A 3.0-A resolution study of nucleotide complexes with aspartate carbamoyltransferase.

The binding sites of CTP, CDP, 5-BrCTP, and ATP to the allosteric site of aspartate carbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) have been found in electron-density maps obtained at about 3 A resolution from x-ray diffraction studies of single crystals. The activator ATP binds in the anti conformation, whereas the inhibitor 5-BrCTP binds in the syn conformation. Both activator and inhibitor bind to the same local region of the enzyme. All of the cytidine nucleotides show important interactions of the base with the protein. The triphosphate conformations are similar, whereas the terminal phosphate of CDP occupies the site of the gamma-phosphate of CTP, thus implying a protein-nucleotide interaction at this site. These results are then related to biochemical studies.

Allosteric Regulation↗