Temperature effects on the allosteric responses of native and chimeric aspartate transcarbamoylases 1 1Edited by J. H. Miller

Abstract

Although structurally very similar, the aspartate transcarbamoylases (ATCase) of Serratia marcescens and Escherichia coli have distinct allosteric regulatory patterns. It has been reported that a S. marcescens chimera, SM : rS5′ec, in which five divergent residues (r93 to r97) of the regulatory polypeptide were replaced with their Escherichia coli counterparts, possessed E. colilike regulatory characteristics. T he reverse chimera EC : rS5′sm, in which the same five residues of E. coli have been replaced with their S. marcescens counterpart, lost both heterotrophic and homotropic responses. These results indicate that the r93-r97 region is critical in defining the ATCase allosteric character. Molecular modeling of the regulatory polypeptides has suggested that the replacement of the S5′ β-strand resulted in disruption of the allosteric-zinc interface. However, the structure-function relationship could be indirect, and the disruption of the interface could influence allostery by altering the global energy of the enzyme. Studies of the temperaturesensitivity of the CTP response demonstrate that it is possible to convert CTP inhibition of the SM : rS5′ec chimera at high temperature to activation below 10 °C. Nonetheless, the temperature response of the native S. marcescens ATCase suggests a strong entropic effect that counteracts the CTP activation. Therefore, it is suggested that the entropy component of the coupling free energy plays a significant role in the determination of both the nature and magnitude of the allosteric effect in ATCase.

Allosteric Signal Transmission Involves Synergy between Discrete Structural Units of the Regulatory Subunit of Aspartate Transcarbamoylase

“…It should be noted that despite the allosteric similarity between the native E. coli and the SM:rS5'ec ATCases, these enzymes were not affected by the V106A and Y77F mutations in a similar manner. This is consistent with previous studies (15) suggesting that the S5′ β-strand is not the sole determinant of the allosteric character and indicates that the hydrophobic interface plays a major role, (iii) Both the ATP-activating and the CTP-inhibiting effects were reduced in the SM: rS5'ec/Y77F and SM:rS5'ec/V106A enzymes relative to the SM:rS5'ec chimera. In this case, it is clear that the allosteric-zinc interface does not discriminate between the ATP and the CTP signals but has an effect on both.…”

“…Site-directed mutagenesis was performed with double-stranded plasmid pPBh601-ec for E. coli (15) and pPBh200-sm for S. marcescens (9) according to the Deng and Nickoloff protocol (19) as modified by Liu et al (10). The single (or double) mutations were generated by incorporating the corresponding mutagenesis primer (or primers) and selection primers with the native plasmid.…”

“…While a high frequency of the amino acid variation has been observed in the r100's and r130's region (9,10), the most significant divergence lies within the S5′ β-strand of the regulatory polypeptide. Interconversions of this S5′ β-strand in the E. coli and S. marcescens ATCases generated chimeric enzymes with significant allosteric disruptions, indicating that the S5′ β-strand played an important role in the determination of the allosteric responses of the native enzymes (10,15).…”

“…The reverse chimera, EC:rS5'sm, has been constructed by replacing the S5′ β-strand of the E. coli enzyme with that of the S. marcescens enzyme. However, the resulting enzyme lost all allosteric responses (15). While these results demonstrate that the nonconserved residues r93-r97 (S5′ β-strand) are critical to establishing the allosteric pattern (10, 15), molecular modeling further suggested that the substitutions of the S5′ β-strand act by perturbing residues at the hydrophobic allosteric-zinc interface (15).…”

“…However, the resulting enzyme lost all allosteric responses (15). While these results demonstrate that the nonconserved residues r93-r97 (S5′ β-strand) are critical to establishing the allosteric pattern (10,15), molecular modeling further suggested that the substitutions of the S5′ β-strand act by perturbing residues at the hydrophobic allosteric-zinc interface (15). This hypothesis is consistent with a series of single amino acid substitutions which indicate that the allosteric-zinc interface can modulate the allosteric responses of the E. coli enzyme.…”