Stabilization of the R Allosteric Structure of Escherichia coli Aspartate Transcarbamoylase by Disulfide Bond Formation

Abstract

Here we report the first use of disulfide bond formation to stabilize the R allosteric structure of Escherichia coli aspartate transcarbamoylase. In the R allosteric state, residues in the 240s loop from two catalytic chains of different subunits are close together, whereas in the T allosteric state they are far apart. By substitution of Ala-241 in the 240s loop of the catalytic chain with cysteine, a disulfide bond was formed between two catalytic chains of different subunits. The cross-linked enzyme did not exhibit cooperativity for aspartate. The maximal velocity was increased, and the concentration of aspartate required to obtain onehalf the maximal velocity, [Asp] 0.5 , was reduced substantially. Furthermore, the allosteric effectors ATP and CTP did not alter the activity of the cross-linked enzyme. When the disulfide bonds were reduced by the addition of 1,4-dithio-DL-threitol the resulting enzyme had kinetic parameters very similar to those observed for the wild-type enzyme and regained the ability to be activated by ATP and inhibited by CTP. Small-angle x-ray scattering was used to verify that the cross-linked enzyme was structurally locked in the R state and that this enzyme after reduction with 1,4-dithio-DL-threitol could undergo an allosteric transition similar to that of the wild-type enzyme. The complete abolition of homotropic and heterotropic regulation from stabilizing the 240s loop in its closed position in the R state, which forms the catalytically competent active site, demonstrates the significance that the quaternary structural change and closure of the 240s loop has in the functional mechanism of aspartate transcarbamoylase.Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) is one of the best characterized allosteric enzymes, so it serves an important role in the study of allosteric regulation. This enzyme catalyzes the committed step of pyrimidine biosynthesis, the carbamoylation of the amino group of L-aspartate by carbamoyl phosphate to form N-carbamoyl-L-aspartate (1). The enzyme demonstrates homotropic cooperativity for the substrate Laspartate and is heterotropically regulated by effectors ATP, CTP (1), and UTP in the presence of CTP (2). Aspartate transcarbamoylase from E. coli is a dodecamer composed of six catalytic chains organized as two trimeric subunits and six regulatory chains organized as three dimeric subunits. Because aspartate transcarbamoylase is so well characterized and serves as a model for allosteric enzymes it is of particular interest to thoroughly understand the molecular mechanism of allostery of this enzyme.Two different structural and functional states of aspartate transcarbamoylase have been characterized (3-6). The low activity, low affinity conformation of the enzyme is called the T state, and the high activity, high affinity conformation of the enzyme is called the R state. The conversion from the T to the R state occurs upon aspartate binding to the holoenzyme in the presence of carbamoyl phosphate, which is the source of the observed homotropic co...

Sequential formation of ion pairs during activation of a sodium channel voltage sensor

“…Our results provide strong support for this model of voltage-dependent gating by demonstrating state-dependent formation of disulfide bonds between cysteine residues substituted for the interacting partners in these ion pairs on the time scale of channel activation and by measuring the coupling energy for interaction of these residues during activation of WT and mutant channels by mutant cycle analysis. Disulfide-bond formation between substituted cysteine residues is a wellestablished method of analysis of the structures of intermediates in protein-folding pathways and of intermediate conformations in allosteric enzymes (21,23,33). Because the reactive sulfhydryls must approach within 2 Å at the time of formation of a disulfide bond, this is a high-resolution method of analysis of intra-protein interactions.…”

A Fluorescent Probe-labeled Escherichia coli Aspartate Transcarbamoylase That Monitors the Allosteric Conformational State

“…Considering that the same reduction in the heterotropic effects of ATP and CTP was observed for the unlabeled C47A/A241C holoenzyme (20), it can be concluded that the reduced effects are because of the mutations and not to the pyrene labeling.…”

“…EK1104 (F Ϫ ara, thi, ⌬(pro-lac), ⌬pyrB, pyrF Ϯ , rpsL) was previously constructed in this laboratory (25). The plasmids pEK613 and pEK614, containing the DNA for the C47A/A241C holoenzyme and C47A/A241C catalytic subunit, respectively, were previously constructed in this laboratory (20).…”

“…Therefore, the 240s loop was chosen as the location for placement of a fluorescent probe that is sensitive to conformation. We chose to conjugate pyrene site specifically to our previously constructed 240s loop mutant, C47A/A241C (20). The substitution of the one naturally occurring cysteine in the catalytic chain, Cys-47, by alanine ensured site-specific labeling.…”

“…1). Previous studies have shown that when Ala-241 in the wild-type enzyme is converted to Cys the modified enzyme has wild-type like properties (20). The fluorophore used in this study, pyrene, fluoresces not only from an excited monomer state, but also from an excited-state dimer, or excimer; excimer fluorescence results from a specific interaction between the excited monomer and a ground-state monomer (21).…”