Complete 1H, 13C, and 15N assignments and secondary structure of the GTPase activating domain of Gs.

Complete 1H, 13C, and 15N assignments for backbone and side-chain atoms of the 145 residue GTPase activating domain of Gs are presented. The combination of gradient-enhanced versions of the HNCACB and CBCA(CO)NNH pulse sequences provided enough information to obtain sequential backbone assignments for residues 2-145 of the polypeptide, as well as assignments of asparagine and glutamine side-chain amides. HBHA(CO)NNH, HCCH-TOCSY, and 13C/15N NOESY-HSQC experiments yielded side-chain 1H and 13C assignments. Chemical shift data and 15N NOESY-HSQC experiments provided information on the secondary structure of the domain, which is similar to that observed in the cognate domain in transducin, a related G protein. The functionally essential C-terminal 15 residues are disordered in solution. These assignments provide a basis for determining the solution structure of the domain.

Separate GTP binding and GTPase activating domains of a G alpha subunit.

Most members of the guanosine triphosphatase (GTPase) superfamily hydrolyze guanosine triphosphate (GTP) quite slowly unless stimulated by a GTPase activating protein or GAP. The alpha subunits (G alpha) of the heterotrimeric G proteins hydrolyze GTP much more rapidly and contain an approximately 120-residue insert not found in other GTPases. Interactions between a G alpha insert domain and a G alpha GTP-binding core domain, both expressed as recombinant proteins, show that the insert acts biochemically as a GAP. The results suggest a general mechanism for GAP-dependent hydrolysis of GTP by other GTPases.

A 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase forms a stable complex with the catalytic subunit leading to markedly altered enzyme activity.

In an effort to clarify effects of specific protein-protein interactions on the properties of the dodecameric enzyme aspartate transcarbamoylase (carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), we initiated studies of a simpler complex containing an intact catalytic trimer and three copies of a fragment from the regulatory chain. The partial regulatory chain was expressed as a soluble 9-kDa zinc-binding polypeptide comprising 11 amino acids encoded by the polylinker of pUC18 fused to the amino terminus of residues 84-153 of the regulatory chain; this polypeptide includes the zinc domain detected in crystallographic studies of the holoenzyme. In contrast to intact regulatory chains, the zinc-binding polypeptide is monomeric in solution because it lacks the second domain responsible for dimer formation and assembly of the dodecameric holoenzyme. The isolated 9-kDa protein forms a tight, zinc-dependent complex with catalytic trimer, as shown by the large shift in electrophoretic mobility of the trimer in nondenaturing polyacrylamide gels. Enzyme assays of the complex showed a hyperbolic dependence of initial velocity on aspartate concentration with Vmax and Km for aspartate approximately 50% lower than the values for free catalytic subunit. A mutant catalytic subunit containing the Lys-164----Glu substitution exhibited a striking increase in enzyme activity at low aspartate concentrations upon interaction with the zinc domain because of a large reduction in Km upon complex formation. These changes in functional properties indicate that the complex of the zinc domain and catalytic trimer is an analog of the high-affinity R ("relaxed") state of aspartate transcarbamoylase, as proposed previously for a transiently formed assembly intermediate composed of one catalytic and three regulatory subunits. Conformational changes at the active sites, resulting from binding the zinc-containing polypeptide chains, were detected by difference spectroscopy with trinitrophenylated catalytic trimers. Isolation of the zinc domain of aspartate transcarbamoylase provides a model protein for study of oligomer assembly, communication between dissimilar polypeptides, and metal-binding motifs in proteins.

Heterotropic effectors promote a global conformational change in aspartate transcarbamoylase.

The sigmoidal dependence of activity on substrate concentration exhibited by the regulatory enzyme aspartate transcarbamoylase (ATCase) of Escherichia coli is generally attributed to a ligand-promoted change in the quaternary structure of the enzyme. Although a global conformational change in ATCase upon the binding of ligands to some of the six active sites is well documented, a corresponding alteration in the structure of the wild-type enzyme upon the addition of the inhibitor, CTP, or the activator, ATP, has not been detected. Such evidence is essential for testing whether heterotropic, as well as homotropic, effects can be accounted for quantitatively in terms of coupled equilibria involving a conformational change in the enzyme and preferential binding of ligands to one conformation or the other. This evidence has now been obtained with a mutant form of ATCase in which Lys 143 in the regulatory chain was replaced by Ala, thereby perturbing interactions at the interface between the regulatory and catalytic chains in the enzyme and destabilizing the low-activity, compact (T) conformation relative to the high-activity, swollen (R) state. Difference sedimentation velocity experiments involving measurements of the changes caused by the binding of the bisubstrate analogue N-(phosphonacetyl)-L-aspartate demonstrated that the sedimentation coefficient of the mutant enzyme was intermediate between that observed for the T and R states of wild-type ATCase. We interpret the results as indicating that the [T]/[R] ratio in phosphate buffer at pH 7.0 is reduced from about 2 X 10(2) for the wild-type enzyme to 2.7 for r143Ala ATCase.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in stability and allosteric properties of aspartate transcarbamoylase resulting from amino acid substitutions in the zinc-binding domain of the regulatory chains.

Changes in subunit interaction energies linked to the allosteric transition of the regulatory enzyme aspartate transcarbamoylase (ATCase; EC 2.1.3.2) from Escherichia coli are localized in part at interfaces between the six catalytic (c) and six regulatory (r) polypeptide chains. Site-directed mutagenesis has been used to construct enzymes with amino acid substitutions in a limited region of the zinc-binding domain of the r chains. Substitution of Ser or His for r114 Cys, one of four cysteines binding the structural zinc ion in the regulatory chain, leads to incorrectly folded chains as shown by the inability to detect stable assembled holoenzyme in cell extracts. Replacement of r111 Asn by Ala at the interface between an r chain and a c chain in the apposing catalytic trimer causes a complete loss of the homotropic and heterotropic effects characteristic of wild-type ATCase. Moreover, sedimentation velocity experiments demonstrated that this mutant enzyme exists in the R ("relaxed") conformation in the absence of active site ligands due to preferential destabilization of the T ("taut") conformation relative to the R state. In contrast, replacement of r113 Asn by Ala at the interface between adjacent r and c chains leads to an increase in the cooperativity of the enzyme. When r139 Lys is replaced by Met, Vmax is reduced by 50% compared to wild-type ATCase, whereas it is increased about 2-fold when r142 Glu is replaced by Asp. Amino acid substitutions in this domain significantly affect subunit interaction energy as measured by rate of subunit exchange when holoenzymes are incubated with isolated catalytic subunits, thus permitting measurements of the effect of the bisubstrate analog N-(phosphonacetyl)-L-asparatate in weakening intersubunit interactions. Subunit exchange increased about 9-fold for the r142 Glu----Asp mutant and almost 20-fold for the r142 Glu----Ala mutant in the presence of the ligand.

Cooperative binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate to aspartate transcarbamoylase and the heterotropic effects of ATP and CTP.

Most investigations of the allosteric properties of the regulatory enzyme aspartate transcarbamoylase (ATCase) from Escherichia coli are based on the sigmoidal dependence of enzyme activity on substrate concentration and the effects of the inhibitor, CTP, and the activator, ATP, on the saturation curves. Interpretations of these effects in terms of molecular models are complicated by the inability to distinguish between changes in substrate binding and catalytic turnover accompanying the allosteric transition. In an effort to eliminate this ambiguity, the binding of the 3H-labeled bisubstrate analog N-(phosphonacetyl)-L-aspartate (PALA) to aspartate transcarbamoylase in the absence and presence of the allosteric effectors ATP and CTP has been measured directly by equilibrium dialysis at pH 7 in phosphate buffer. PALA binds with marked cooperativity to the holoenzyme with an average dissociation constant of 110 nM. ATP and CTP alter both the average affinity of ATCase for PALA and the degree of cooperativity in the binding process in a manner analogous to their effects on the kinetic properties of the enzyme; the average dissociation constant of PALA decreases to 65 nM in the presence of ATP and increases to 266 nM in the presence of CTP while the Hill coefficient, which is 1.95 in the absence of effectors, becomes 1.35 and 2.27 in the presence of ATP and CTP, respectively. The isolated catalytic subunit of ATCase, which lacks the cooperative kinetic properties of the holoenzyme, exhibits only a very slight degree of cooperativity in binding PALA. The dissociation constant of PALA from the catalytic subunit is 95 nM. Interpretation of these results in terms of a thermodynamic scheme linking PALA binding to the assembly of ATCase from catalytic and regulatory subunits demonstrates that saturation of the enzyme with PALA shifts the equilibrium between holoenzyme and subunits slightly toward dissociation. Ligation of the regulatory subunits by either of the allosteric effectors leads to a change in the effect of PALA on the association-dissociation equilibrium.