|Title||Subsite interactions of ribonuclease T1: Asn36 and Asn98 accelerate GpN transesterification through interactions with the leaving nucleoside N.|
|Publication Type||Journal Article|
|Year of Publication||1991|
|Authors||Steyaert, J., A. F. Haikal, L. Wyns, and P. Stanssens|
|Date Published||1991 Sep 3|
|Keywords||Amino Acid Sequence, Asparagine, Base Composition, Binding Sites, Dinucleoside Phosphates, Esterification, Guanosine Monophosphate, Kinetics, Protein Conformation, Ribonuclease T1, Substrate Specificity|
We previously presented evidence that ribonuclease T1 (RNase T1; EC 22.214.171.124) contains a subsite that, by interacting with the leaving nucleoside N of GpN dinucleoside phosphate substrates, contributes to catalysis. The kcat values for transphosphorylation follow the order GpC greater than GpA greater than GpU whereas the equilibrium dissociation constants for these substrates are very similar [Steyaert, J., Wyns, L., & Stanssens, P. (1991) Biochemistry (preceding paper in this issue)]. Consistent with this notion, we find that the rate of transesterification of the synthetic substrate GpMe, in which the leaving nucleoside is replaced by a methanol group, is at least 3 orders of magnitude lower than that of GpN substrates. The enzyme's affinity for GpMe is very similar to that for the various GpN substrates, indicating that the apparent contribution of the leaving nucleoside to ground-state binding is minimal. To identify the side chains that belong to the RNase T1 subsite, we searched for amino acid substitutions that differentially affect the transesterification kinetics of GpNs versus GpMe. The Asn36Ala, Tyr38Phe, His92Gln, and Asn98Ala mutants have been analyzed. Of these, the Asn36Ala and Asn98Ala substitutions reduce the transphosphorylation rate of the different GpNs considerably whereas they have virtually no effect on the rate of GpMe transphosphorylation. This observation shows that the Asn36 and Asn98 amide functions are part of the RNase T1 subsite. The sum of the contributions of the two residues accounts quite precisely for the differences in turnover rates among GpC, GpA, and GpU.