Hydrophobic effects on protein/nucleic acid interaction: enhancement of substrate binding by mutating tyrosine 45 to tryptophan in ribonuclease T1.
Hydrophobic effects on binding of ribonuclease T1 to guanine bases of several ribonucleotides have been proved by mutating a hydrophobic residue at the recognition site and by measuring the effect on binding. Mutation of a hydrophobic surface residue to a more hydrophobic residue (Tyr45----Trp) enhances the binding to ribonucleotides, including mononucleotide inhibitor and product, and a synthetic substrate-analog trinucleotide as well as the binding to dinucleotide substrates and RNA. Enhancements on binding to non-substrate ribonucleotides by the mutation have been observed with free energy changes ranging from -2.2 to -3.9 kJ/mol. These changes are in good agreement with that of substrate binding, -2.3 kJ/mol, which is calculated from Michaelis constants obtained from kinetic studies. It is shown, by comparing the observed and calculated changes in binding free energy with differences in the observed transfer free energy changes of the amino acid side chains from organic solvents to water, that the enhancement observed on guanine binding comes from the difference in the hydrophobic effects of the side chains of tyrosine and tryptophan. Furthermore, a linear relationship between nucleolytic activities and hydrophobicity of the residues (Ala, Phe, Tyr, Trp) at position 45 is observed. The mutation could not change substantially the base specificity of RNase T1, which exhibits a prime requirement for guanine bases of substrates.
Hakoshima
T
T
Faculty of Pharmaceutical Sciences, Osaka University, Japan.
Tanaka
M
M
Itoh
T
T
Tomita
K I
KI
Amisaki
T
T
Nishikawa
S
S
Morioka
H
H
Uesugi
S
S
Ohtsuka
E
E
Ikehara
M
M
D001665:Binding Sites; D006157:Guanosine Monophosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009843:Oligoribonucleotides / Q000378:metabolism; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000235:genetics*; D012265:Ribonucleotides / Q000378:metabolism*; D013499:Surface Properties; D014364:Tryptophan / Q000737:chemistry
D009843:Oligoribonucleotides; D012265:Ribonucleotides; C026693:2'-guanylic acid; C073400:adenyl-(3',5')-2'-deoxyfluoroguanylyl-(3',5')-adenosine; D012313:RNA; C068152:3'-guanylic acid; D006157:Guanosine Monophosphate; D014364:Tryptophan; D006163:Ribonuclease T1