title: Modes of mononucleotide binding to ribonuclease T1.
abstract: The binding of the mononucleotide inhibitors 2'-GMP, 3'-GMP, and 5'-GMP to genetically engineered ribonuclease T1 has been investigated by conventional inhibition kinetics, fluorimetric titrations, molecular modeling, and fast relaxation techniques. The fluorimetric titrations in conjunction with molecular modeling revealed that apart from the already known primary binding site, three to four additional sites are present on the enzyme's surface. The association constants obtained from the fluorimetric titrations and the temperature jump experiments range between 3.1 x 10(6) M-1 and 4.3 x 10(6) M-1, indicating that the binding of the mononucleotides to the specific binding site of ribonuclease T1 is at least one order of magnitude tighter than has been anticipated so far. The kinetics of binding are nearly diffusion controlled with a kon determined for 2'-GMP and 3'-GMP, as (5.0 +/- 0.5 x 10(9) and 6.1 +/- 0.5 x 10(9) M-1, s-1 and koff as 1.2 +/- 0.2 x 10(3) and 2.0 +/- 0.3 x 10(3) s-1, respectively. Molecular modeling studies indicate that all three nucleotides are able to bind via their phosphate group to a positively charged array of surface amino acids including His27, His40, Lys41, and most probably Lys25 without obvious stereochemical hindrance. We propose that RNA wraps around RNase T1 in a similar fashion via phosphate binding when enzymatic hydrolysis occurs.
authors.affiliation: Institut für Kristallographie, Freie Universität Berlin, Federal Republic of Germany.
authors.forename: Y
authors.initials: Y
authors.lastname: Georgalis
authors.forename: A
authors.initials: A
authors.lastname: Zouni
authors.forename: P
authors.initials: P
authors.lastname: Zielenkiewicz
authors.forename: J F
authors.initials: JF
authors.lastname: Holzwarth
authors.forename: R
authors.initials: R
authors.lastname: Clarke
authors.forename: U
authors.initials: U
authors.lastname: Hahn
authors.forename: W
authors.initials: W
authors.lastname: Saenger
mesh_terms: D001665:Binding Sites; D003198:Computer Simulation; D006157:Guanosine Monophosphate / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D006163:Ribonuclease T1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: C026693:2'-guanylic acid; C068152:3'-guanylic acid; D006157:Guanosine Monophosphate; D006163:Ribonuclease T1