Modes of modifier action in E. coli aspartate transcarbamylase

Wedler, Frederick C.; Gasser, Frank J.

doi:10.1016/0003-9861(74)90455-x

Cited by 30 publications

(14 citation statements)

References 41 publications

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“…3). Although there is a suggestion of a discontinuity at 200C, which would be consistent with the thermal transition reported by Wedler and Gasser (19,20), the data were fit to the polynomial AH = A + B(T -15) + C(T -15)2. [3] Contributions of the buffers were estimated from values for AHln.…”

Section: Resultsmentioning

confidence: 99%

Thermodynamics of assembly of Escherichia coli aspartate transcarbamoylase.

McCarthy¹,

Allewell²

1983

Proc. Natl. Acad. Sci. U.S.A.

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Reaction microcalorimetry and potentiometry have been used to define the thermodynamics of assembly of Escherichia coli aspartate transcarbamoylase (aspartate carbamoyltransferase, carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) from its catalytic and regulatory subunits and the linkage between assembly and proton binding. Over the pH range 7-9.5 and the temperature range 15-300C, assembly is characterized by negative enthalpy and heat capacity changes and positive entropy changes. The dependence of the enthalpy and entropy changes on pH is complex; however, the negative heat capacity change results in both quantities becoming more negative with increasing temperature. Assembly is linked to the binding of protons; the effects observed can be fit to models involving six or more ionizable groups with pK values of 7. 3-7.4, 8.5-8.8, and 9.2-9.5, which ionize cooperatively. Contributions from additional groups cannot be ruled out and are in fact expected. The overall pattern of thermodynamic effects implies a complex set of intersubunit interactions. Protonation reactions and increased hydrogen bonding are likely to be the major sources of the negative enthalpy change; however, the negative heat capacity change results primarily from changes in solvent structure associated with hydrophobic and electrostatic bond formation with changes in lowfrequency vibrational modes making a secondary contribution. Similarly, the relatively small entropy change observed within the temperature range examined probably reflects the balance between positive contributions from increased hydrophobic and electrostatic bonding and negative contributions from increased hydrogen bonding and damping of low-frequency vibrational modes. cial from a mechanistic point of view will require additional information (9-12).Because allosteric regulation is a thermodynamic phenomenon, thermodynamic information will clearly be essential. Allewell and co-workers have examined the energy changes associated with binding of substrate analogs (7,13) and NTPs (14) and found that binding of both types of ligand is linked thermodynamically to binding of protons. It has also been shown that the nature of this linkage is different for the activator, ATP, and the inhibitor, CTP (14).We have used reaction microcalorimetry to measure, over a range of pH and temperature, the enthalpy changes that accompany assembly of the native enzyme from isolated subunits in the reaction ,2c3 + 3 r2 W6, where C3 and r2 represent catalytic and regulatory subunits, respectively. Linked proton binding was characterized directly by potentiometry. These data together with estimates of the free energy of the c-r interactions from the literature allow the energy changes associated with assembly to be calculated as a function of both pH and temperature. Hence they provide thermodynamic criteria that can be used to test and develop models of the allosteric mechanism. METHODSProteins. The native enzyme was purified from a derepressed diploid strain of E. coli as...

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Section: Resultsmentioning

confidence: 99%

Thermodynamics of assembly of Escherichia coli aspartate transcarbamoylase.

McCarthy¹,

Allewell²

1983

Proc. Natl. Acad. Sci. U.S.A.

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“…A similar behaviour has been observed in the case of ~-glyceraldehyde-3-phosphate dehydrogenase from a thermophilic archaeobacterium (Fabry & Hensel, 1987). Interestingly, such a break in the Arrhenius plot has also been reported in the case of E. coli ATCase (Wedler & Gasser, 1974), but at a lower temperature. The substantial decrease of the enthalpy of activation and of the negative value of entropy of activation when the temperature is raised from 37 "C to 96 "C indicates that the transition state of the reaction is reached more easily at high temperature.…”

Section: P Al?_yssimentioning

confidence: 91%

“…This procedure was used taking into account the behaviour of E. coli ATCase. This homologous enzyme operates through an ordered mechanism in which CP binds first, followed by aspartate (Porter e t a/., 1969;Schaffer & Stark, 1972;Wedler & Gasser, 1974;Issaly et al, 1982;Hsuanyu & Wedler, 1987;Parmentier et al, 1992). As a consequence, the CP saturation curve is sigmoidal only in the presence of a high concentration of aspartate and the apparent cooperativity for CP reflects only that for aspartate (England e t al., 1994).…”

Section: Catalytic Properties Of P Abyssi Atcasementioning

confidence: 99%

The catalytic and regulatory properties of aspartate transcarbamoylase from Pyrococcus abyssi, a new deep-sea hyperthermophilic archaeobacterium

et al. 1994

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“…1 to 3; Table 2). To understand the regulatory logic of ATCase, one must always consider the competition of ATP and CTP for the same allosteric binding sites (10,11,17), whether the two nucleotides produce different heterotropic responses (8,17) or not (2,7,19). As observed in these studies, ATP-activated catalysis was reduced in the presence of CTP regardless of whether CTP functioned independently as an inhibitor or as an activator or had no effect on the enzyme.…”

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confidence: 92%

ATP-liganded form of aspartate transcarbamoylase, the logical regulatory target for allosteric control in divergent bacterial systems

1988

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In Escherichia coli, the mechanism for regulatory control of aspartate transcarbamoylase is clear; CTP allosterically inhibits catalysis in direct competition with ATP. However, both CTP and ATP may be activators or may have no effect on aspartate transcarbamoylases from other enteric bacteria. A common regulatory logic observed was that the ATP-activated enzymes were rendered less active as the result of competition with CTP, regardless of the independent effects.

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Modes of modifier action in E. coli aspartate transcarbamylase

Cited by 30 publications

References 41 publications

Thermodynamics of assembly of Escherichia coli aspartate transcarbamoylase.

Thermodynamics of assembly of Escherichia coli aspartate transcarbamoylase.

The catalytic and regulatory properties of aspartate transcarbamoylase from Pyrococcus abyssi, a new deep-sea hyperthermophilic archaeobacterium

ATP-liganded form of aspartate transcarbamoylase, the logical regulatory target for allosteric control in divergent bacterial systems

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