title: Dissection of the ribonuclease T1 subsite. The transesterification kinetics of Asn36Ala and Asn98Ala ribonuclease T1 for minimal dinucleoside phosphates.
abstract: Ribonuclease T1 contains a subsite which by interacting with the leaving nucleoside N of GpN dinucleoside phosphate substrates, contributes to catalysis [Steyaert, J., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8661-8665]. The Asn36Ala and Asn98Ala mutations reduce the transesterification rates of GpA, GpC and GpU considerably whereas they have virtually no effect on the transesterification kinetics of the synthetic substrate guanosine 3'-(methyl phosphate) (GpMe) (in which the leaving nucleoside is replaced by methanol), indicating that the Asn36 and Asn98 side chains are part of the RNase T1 subsite [Steyaert, J., Haikal, A. F., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8666-8670]. The kinetics of the Asn36Ala, Asn98Ala and wild-type catalyzed transesterification of guanosine 3'-(5'-D-ribosyl phosphate) (GpRib), another GpN analog in which the leaving groups is replaced by D-ribose, enables the mapping of the subsite interactions provided by Asn36 and Asn98. We find that the Asn36 amide function contributes 4.6 kJ/mol to catalysis through interactions with the ribose moiety of the leaving nucleoside. Asn98 is at least in part responsible for the subsite preference for cytidine; the Asn98 side chain preferentially binds cytosine as the leaving nucleoside base.
authors.affiliation: Vrije Universiteit Brussel, Instituut Moleculaire Biologie, St.-Genesius-Rode, Belgium.
authors.forename: J
authors.initials: J
authors.lastname: Steyaert
authors.forename: A F
authors.initials: AF
authors.lastname: Haikal
authors.forename: P
authors.initials: P
authors.lastname: Stanssens
authors.forename: L
authors.initials: L
authors.lastname: Wyns
mesh_terms: D000409:Alanine / Q000235:genetics*; D001216:Asparagine / Q000235:genetics*; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D004734:Energy Metabolism; D004952:Esters / Q000378:metabolism; D007700:Kinetics; D006163:Ribonuclease T1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D015226:Dinucleoside Phosphates; D004952:Esters; D001216:Asparagine; D006163:Ribonuclease T1; D000409:Alanine