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

Nm, Allwell; Ge, Hofmann; Zaug, Arthur J.; Lennick, Michael

doi:10.1021/bi00581a016

Cited by 21 publications

(18 citation statements)

References 39 publications

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“…Therefore, the wild-type enzyme is stabilizing the bound PALA as the dianion at both pH values, whereas the mutant is stabilizing the bound PALA as the monoanion also at both pH values. The observation that PALA binds to the enzyme as the dianion at pH 7.0 confirms previous experiments by NMR (Cohen & Schachman, 1986) and potentiometry (Allewell et al, 1979). The above experiments demonstrate that the replacement of Arg-54 by an alanine dramatically alters the pK, of the phosphonate group of PALA in the enzyme-ligand complex.…”

Section: The Effective Ionic Environment Of the Arg-54 -+ Ala And Wilsupporting

confidence: 89%

Arginine 54 in the active site of escherichia coli aspartate transcarbamoylase is critical for catalysis: A site‐specific mutagenesis, NMR, and X‐ray crystallographic study

Stebbins¹,

Robertson²,

Roberts³

et al. 1992

Protein Science

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The replacement of Arg-54 by Ala in the active site of Escherichia coli aspartate transcarbamoylase causes a 17,000-fold loss of activity but does not significantly influence the binding of substrates or substrate analogs (Stebbins, J.W., Xu, W., , Biochemistry 28, 2592-2600. In the X-ray structure of the wild-type enzyme, Arg-54 interacts with both the anhydride oxygen and a phosphate oxygen of carbamoyl phosphate (CP) (Gouaux, J.E. & Lipscomb, W.N., 1988, Proc. Natl. Acad. Sci. USA 85,4205-4208). The Arg-54 Ala enzyme was crystallized in the presence of the transition state analog N-phosphonoacetyl-L-aspartate (PALA), data were collected to a resolution limit of 2.8 A, and the structure was solved by molecular replacement. The analysis of the refined structure (R factor = 0.18) indicates that the substitution did not cause any significant alterations to the active site, except that the side chain of the arginine was replaced by two water molecules. 31P-NMR studies indicate that the binding of CP to the wild-type catalytic subunit produces an upfield chemical shift that cannot reflect a significant change in the ionization state of the CP but rather indicates that there are perturbations in the electronic environment around the phosphate moiety when CP binds to the enzyme. The pH dependence of this upfield shift for bound CP indicates that the catalytic subunit undergoes a conformational change with a pK, -7.7 upon CP binding. Furthermore, the linewidth of the 31P signal of CP bound to the Arg-54 +Ala enzyme is significantly narrower than that of CP bound to the wild-type catalytic subunit at any pH, although the change in chemical shift for the C P bound to the mutant enzyme is unaltered. 3'P-NMR studies of PALA complexed to the wild-type catalytic subunit indicate that the phosphonate group of the bound PALA exists as the dianion at pH 7.0 and 8.8, whereas in the Arg-54 -t Ala catalytic subunit the phosphonate group of the bound PALA exists as the monoanion at pH 7.0 and 8.8. Thus, the side chain of Arg-54 is essential for the proper ionization of the phosphonate group of PALA and by analogy the phosphate group in the transition state. These data support the previously proposed proton transfer mechanism, in which a fully ionized phosphate group in the transition state accepts a proton during catalysis.Keywords: allosteric enzyme; aspartate carbamoyltransferase; enzyme mechanism; mutant structure; N-phosphonoacetyl-L-aspartate; 31P-NMR; site-specific mutagenesis

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Section: The Effective Ionic Environment Of the Arg-54 -+ Ala And Wilsupporting

confidence: 89%

Arginine 54 in the active site of escherichia coli aspartate transcarbamoylase is critical for catalysis: A site‐specific mutagenesis, NMR, and X‐ray crystallographic study

Stebbins¹,

Robertson²,

Roberts³

et al. 1992

Protein Science

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“…The product of the dissociation under pressure is shown to be the monomer by its 30 ns rotational relaxation time and by the uniform appearance in time of the three mixed isozymes when the M4 and H4 lactate dehydrogenases are jointly subjected to high pressure (4). Fig 1 shows the degree of dissociation, calculated from the tryptophan fluorescence polarization, observed at pressures of up to 2.5 kbar, at three protein concentrations.…”

Section: Resultsmentioning

confidence: 99%

Mapping Structural Perturbations in Escherichia Coli Aspartate Transcarbamylase by Medium Resolution Hydrogen Exchange

Burz¹,

Allewell²

1986

Biophysical Journal

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“…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).…”

mentioning

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

Cited by 21 publications

References 39 publications

Arginine 54 in the active site of escherichia coli aspartate transcarbamoylase is critical for catalysis: A site‐specific mutagenesis, NMR, and X‐ray crystallographic study

Arginine 54 in the active site of escherichia coli aspartate transcarbamoylase is critical for catalysis: A site‐specific mutagenesis, NMR, and X‐ray crystallographic study

Mapping Structural Perturbations in Escherichia Coli Aspartate Transcarbamylase by Medium Resolution Hydrogen Exchange

Thermodynamics of assembly of Escherichia coli aspartate transcarbamoylase.

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