Abstract
A hybrid version of Escherichia coli aspartate transcarbamoylase was investigated in which one catalytic subunit has the wild-type sequence, and the other catalytic subunit has Glu-239 replaced by Gln. Since Glu-239 is involved in intersubunit interactions, this hybrid could be used to evaluate the extent to which T state stabilization is required for homotropic cooperativity and for heterotropic effects. Reconstitution of the hybrid holoenzyme (two different catalytic subunits with three wild-type regulatory subunits) was followed by separation of the mixture by anion-exchange chromatography. To make possible the resolution of the three holoenzyme species formed by the reconstitution, the charge of one of the catalytic subunits was altered by the addition of six aspartic acid residues to the C terminus of each of the catalytic chains (AT-C catalytic subunit). Control experiments indicated that the AT-C catalytic subunit as well as the holoenzyme formed with AT-C and wild-type regulatory subunits had essentially the same homotropic and heterotropic properties as the native catalytic subunit and holoenzyme, indicating that the addition of the aspartate tail did not influence the function of either enzyme. The control reconstituted holoenzyme, in which both catalytic subunits have Glu-239 replaced by Gln, exhibited no cooperativity, an enhanced affinity for aspartate, and essentially no heterotropic response identical to the enzyme isolated without reconstitution. The hybrid containing one normal and one mutant catalytic subunit exhibited homotropic cooperativity with a Hill coefficient of 1.4 and responded to the nucleotide effectors at about 50% of the level of the wild-type enzyme. Small angle x-ray scattering experiments with the hybrid enzyme indicated that in the absence of ligands it was structurally similar, but not identical, to the T state of the wild-type enzyme. In contrast to the wild-type enzyme, addition of carbamoyl phosphate induced a significant alteration in the scattering pattern, whereas the bisubstrate analog N-phosphonoacetyl-L-aspartate induced a significant change in the scattering pattern indicating the transition to the R-structural state. These data indicate that in the hybrid enzyme only three of the usual six interchain interactions involving Glu-239 are sufficient to stabilize the enzyme in a low affinity, low activity state and allow an allosteric transition to occur.
Highlights
¶ To whom reprint requests should be addressed: Dept. of Chemistry, Boston College, Merkert Chemistry Center, Chestnut Hill, MA 02467
The active sites are located at the interfaces between the catalytic chains, and the nucleotide effectors bind to the same site on each of the regulatory chains [5,6,7,8,9]
Aspartate saturation curves were determined for the wild-type, the at concentrations close to those of (AT-C), and the E239Q catalytic subunits, and the kinetic parameters were calculated from these curves (Table I)
Summary
Materials—Agarose, ATP, CTP, L-aspartate, N-carbamoyl-L-aspartate, potassium dihydrogen phosphate, and uracil were obtained from Sigma. The DNA fragment containing the modified pyrB gene was mixed with the linear T-tail vector, pGEM-T, and treated with T4 DNA ligase for 16 h at 4 °C Both a candidate with the PCR product inserted and the plasmid used for expression, pEK54 [17], were digested with the restriction enzymes BstEII and SacI. Formation and Purification of Reconstituted Mutant Holoenzymes— Equal amounts of purified E239Q-C catalytic subunit and AT-C catalytic subunit were mixed with excess regulatory subunit and dialyzed overnight against 50 mM Tris acetate buffer, pH 8.3, 2 mM 2-mercaptoethanol, and 0.1 mM zinc acetate [25]. The enzyme solution was diluted so all the scattering curves were performed at the identical protein concentration These data were determined from the aspartate saturation curves. Colorimetric assays were performed at 25 °C in 50 mM Tris acetate buffer, pH 8.3, and saturating levels of carbamoyl phosphate (4.8 mM)
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