240s Loop Interactions Stabilize the T State of Escherichia coli Aspartate Transcarbamoylase

Abstract

Here the functional and structural importance of interactions involving the 240s loop of the catalytic chain for the stabilization of the T state of aspartate transcarbamoylase were tested by replacement of Lys-244 with Asn and Ala. For the K244A and K244N mutant enzymes, the aspartate concentration required to achieve halfmaximal specific activity was reduced to 8.4 and 4.0 mM, respectively, as compared with 12.4 mM for the wild-type enzyme. Both mutant enzymes exhibited dramatic reductions in homotropic cooperativity and the ability of the heterotropic effectors to modulate activity. Small angle x-ray scattering studies showed that the unligated structure of the mutant enzymes, and the structure of the mutant enzymes ligated with N-phosphonacetyl-L-aspartate, were similar to that observed for the unligated and N-phosphonacetyl-L-aspartateligated wild-type enzyme. A saturating concentration of carbamoyl phosphate alone has little influence on the small angle x-ray scattering of the wild-type enzyme. However, carbamoyl phosphate was able to shift the structure of the two mutant enzymes dramatically toward R, establishing that the mutations had destabilized the T state of the enzyme. The x-ray crystal structure of K244N enzyme showed that numerous local T state stabilizing interactions involving 240s loop residues were lost. Furthermore, the structure established that the mutation induced additional alterations at the subunit interfaces, the active site, the relative position of the domains of the catalytic chains, and the allosteric domain of the regulatory chains. Most of these changes reflect motions toward the R state structure. However, the K244N mutation alone only changes local conformations of the enzyme to an R-like structure, without triggering the quaternary structural transition. These results suggest that loss of cooperativity and reduction in heterotropic effects is due to the dramatic destabilization of the T state of the enzyme by this mutation in the 240s loop of the catalytic chain.Regulation of metabolic pathways via enzymes whose activity is dependent upon the concentration of various metabolites is of paramount importance for all living systems. As a response to a change in the concentration of certain metabolites, a regulatory enzyme can alter its catalytic activity thereby regulating the flux of metabolic intermediates. In particular, a change in function (such as the affinity for substrate) necessarily mandates a change in structure (1). One excellent example of a regulatory enzyme in which there are dramatic functional and structural changes dependent upon the concentrations of substrates and allosteric effectors is Escherichia coli aspartate transcarbamoylase.Aspartate transcarbamoylase (EC 2.1.3.2) catalyzes the first reaction in pyrimidine biosynthesis, the reaction between carbamoyl phosphate and L-aspartate to form N-carbamoyl-L-aspartate and inorganic phosphate. The enzyme shows homotropic cooperativity for the substrate L-aspartate (2). Its activity is inhibited heterotropically by...

Computational Studies of Tryptophanyl-tRNA Synthetase: Activation of ATP by Induced-Fit

“…22 The PreTS conformation is stable only in the presence of Mg 2+ ·ATP (and Trp), suggesting that it is comparable to the "relaxed" states of well-studied allosteric proteins like hemoglobin, 23,24 aspartate carbamoyl transferase, [25][26][27][28][29] and glycogen phosphorylase. 30,31 In keeping with this notion, interactions between TrpRS and ATP are far more optimal in the PreTS state than in the Open state.…”

The first high pH structure of Escherichia coli aspartate transcarbamoylase

“…The unit cell dimensions of the crystals of ATCase grown at pH 8.5, in the H3 space group, T high_apo , ( a = b = 129.78 Å, c = 197.74 Å) are similar to the unit cell dimensions of the few other T‐state crystals of ATCase in the H3 space group. Two of these structures in the H3 space group are of the wild‐type enzyme with substrates or substrate analogs in the active site (PDB entries: 1ROC24 and 2AIR40), whereas two were for the P268A (PDB entry 1EZZ41) and the K244N (PDB entry 1SKU32) mutant versions of ATCase in the absence of substrates or substrate analogues. The similarity of the unit cell dimensions of the T high_apo crystals reported here, as compared with these known T‐state structures in the same space group grown at pH values of seven or less, suggested that the crystals grown at pH 8.5 were in the T‐quaternary structure.…”

“…The similarity of the unit cell dimensions of the T high_apo crystals reported here, as compared with these known T‐state structures in the same space group grown at pH values of seven or less, suggested that the crystals grown at pH 8.5 were in the T‐quaternary structure. Therefore, the initial model used for the refinement of the structure was the T‐state structure of the K244N mutant ATCase in the H3 space group (PDB code 1SKU) 32. The data for the T high_apo were refined to a R factor and R free of 21.5% and 24.7%, respectively, with 213 water molecules in the asymmetric unit.…”

“…The initial model for the high pH structure was derived from the coordinates of a mutant version of ATCase (K244N) (PDB code 1SKU) which crystallized at pH 7 in the same space group (H3) and with similar unit cell dimensions ( a = b = 125.67 c = 198.20) 32. Before refinement with CNS,33 all the waters and ligands were removed.…”

Comparison of two T-state structures of regulatory-chain mutants ofEscherichia coliaspartate transcarbamoylase suggests that His20 and Asp19 modulate the response to heterotropic effectors

“…5a and 5b). The importance of this interaction for the allosteric transition has been evaluated and described previously (Alam et al, 2004).…”