Aspartate transcarbamylase of Escherichia coli. Heterogeneity of binding sites for carbamyl phosphate and fluorinated analogs of carbamyl phosphate.

Ridge, John A.; Roberts, Frank M.; Schäffer, Michael; Stark, George R.

doi:10.1016/s0021-9258(17)33046-6

Cited by 16 publications

(5 citation statements)

References 44 publications

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“…If this model is correct, our reported values for Ai>H+ and AFfEnding for succinate would include a contribution from the second 3 mol of CP bound. However, Ridge et al (1976), who also examined this question, came to the conclusion that c6r6, when prepared from E. coli grown in the original Gerhart & Holoubek (1967) medium (as was the case for the protein used in this study), is able to bind 5.4 mol of CP in the absence of succinate. Although we have no direct binding data for our protein, we have chosen to interpret the data in terms of the simplest possible model:…”

Section: Resultsmentioning

confidence: 85%

Bohr effect of Escherichia coli aspartate transcarbamylase. Linkages between substrate binding, proton binding, and conformational transitions

Nm¹,

Ge²,

Zaug³

et al. 1979

Biochemistry

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Section: Resultsmentioning

confidence: 85%

Bohr effect of Escherichia coli aspartate transcarbamylase. Linkages between substrate binding, proton binding, and conformational transitions

Nm¹,

Ge²,

Zaug³

et al. 1979

Biochemistry

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“…The theoretical curves computed in this manner do not differ appreciably from those shown in Figures 1 -3 (similar conclusions apply for the theoretical curves in Figure 4). 7 6 Many studies have been performed on the affinity of ATCase and of the isolated subunits for a variety of ligands such as CTP, ATP, CbmP, and succinate Hammes et al, 1970;Gray et al, 1973;Matsumoto and Hammes, 1973;Rosenbusch and Griffin, 1973;Ridge et al, 1976;Suter and Rosenbusch, 1976a,b). The pattern of binding is complex and both positive and negative cooperativity have been observed.…”

Section: Discussionmentioning

confidence: 99%

“…Some of the discrepancies may be attributable to technical difficulties inherent in the measurement of binding of a ligand like CbmP which decomposes during the experiment or a ligand like succinate which is so weakly bound. In addition very recent studies (Ridge et al, 1976) have shown that differences in the preparations of the enzyme (or subunits) may be responsible for the variations in binding data.…”

Section: Discussionmentioning

confidence: 99%

Allosteric regulation of aspartate transcarbamoylase. Analysis of the structural and functional behavior in terms of a two-state model

Howlett¹,

Blackburn²,

Compton³

et al. 1977

Biochemistry

142

100

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The kinetic and physical properties of the allosteric enzyme, aspartate transcarbamoylase from Escherichia coli, have been analyzed according to the two-state model (Monod, J., Wyman, J., and Changeux, J.-P. (1965), J . Mol. Biol. 12, 88). An internally consistent set of calculated parameters accounted quantitatively for the results from diverse experiments including: (a) enzyme kinetics as a function of the concentration of aspartate in the presence of saturating carbamoyl phosphate; (b) gross conformational changes of the enzyme, revealed by the decrease in the sedimentation coefficient and the increase in the reactivity of the sulfhydryl groups of the regulatory subunits, as a function of the extent of saturation of the active sites by the bisubstrate analogue, N-(phosphonacety1)-L-aspartate; (c) stimulation of enzymic activity at low concentrations of the substrate analogue, succinate; and (d) effects of the inhibitor, CTP, and the activator, ATP, on the kinetic and physical properties of the enzyme. The data were interpreted in terms of an equilibrium between a constrained or low-affinity (T) state and a relaxed or highaffinity (R) form of the enzyme and the perturbation of the equilibrium by the addition of various ligands. In the absence

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“…2-HEP and HMP were synthesized as previously reported . The following substrate analogues were synthesized according to literature procedures: 2- 18 OH-HEP, (2 R )- and (2 S )-HPP, 3-hydroxypropylphosphonate, 2-hydroxybutylphosphonate, 3-fluoro-2-hydroxypropylphosphonate, 2-fluoroethylphosphonate, phosphonoacetaldehyde, and 1-hydroxyethylphosphonate . Ethylphosphonate and aminoethylphosphonate were purchased from Sigma-Aldrich.…”

Section: Methodsmentioning

confidence: 99%

Hydroperoxylation by Hydroxyethylphosphonate Dioxygenase

2009

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Hydroxyethylphosphonate dioxygenase (HEPD) catalyzes the O2-dependent cleavage of the carbon−carbon bond of 2-hydroxyethylphosphonate (2-HEP) to afford hydroxymethylphosphonate (HMP) and formate without input of electrons or use of any organic cofactors. Two mechanisms have been proposed to account for this reaction. One involves initial hydroxylation of substrate to an acetal intermediate and its subsequent attack onto an Fe(IV)-oxo species. The second mechanism features initial hydroperoxylation of substrate followed by a Criegee rearrangement. To distinguish between the two mechanisms, substrate analogues were synthesized and presented to the enzyme. Hydroxymethylphosphonate was converted into phosphate and formate, and 1-hydroxyethylphosphonate was converted to acetylphosphate, which is an inhibitor of the enzyme. These results provide strong support for a Criegee rearrangement with a phosphorus-based migrating group and require that the O−O bond of molecular oxygen is not cleaved prior to substrate activation. (2R)-Hydroxypropylphosphonate partitioned between conversion to 2-oxopropylphosphonate and hydroxymethylphosphonate, with the latter in turn converted to phosphate and formate. Collectively, these results support a mechanism that proceeds by hydroperoxylation followed by a Criegee rearrangement.

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Aspartate transcarbamylase of Escherichia coli. Heterogeneity of binding sites for carbamyl phosphate and fluorinated analogs of carbamyl phosphate.

Cited by 16 publications

References 44 publications

Bohr effect of Escherichia coli aspartate transcarbamylase. Linkages between substrate binding, proton binding, and conformational transitions

Bohr effect of Escherichia coli aspartate transcarbamylase. Linkages between substrate binding, proton binding, and conformational transitions

Allosteric regulation of aspartate transcarbamoylase. Analysis of the structural and functional behavior in terms of a two-state model

Hydroperoxylation by Hydroxyethylphosphonate Dioxygenase

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