Computer modeling studies on the binding of 2',5'-linked dinucleoside phosphates to ribonuclease T1-influence of subsite interactions on the substrate specificity.
The modes of binding of Gp(2',5')A, Gp(2',5')C, Gp(2',5')G and Gp(2',5')U to RNase T1 have been determined by computer modelling studies. All these dinucleoside phosphates assume extended conformations in the active site leading to better interactions with the enzyme. The 5'-terminal guanine of all these ligands is placed in the primary base binding site of the enzyme in an orientation similar to that of 2'-GMP in the RNase T1-2'-GMP complex. The 2'-terminal purines are placed close to the hydrophobic pocket formed by the residues Gly71, Ser72, Pro73 and Gly74 which occur in a loop region. However, the orientation of the 2'-terminal pyrimidines is different from that of 2'-terminal purines. This perhaps explains the higher binding affinity of the 2',5'-linked guanine dinucleoside phosphates with 2'-terminal purines than those with 2'-terminal pyrimidines. A comparison of the binding of the guanine dinucleoside phosphates with 2',5'- and 3',5'-linkages suggests significant differences in the ribose pucker and hydrogen bonding interactions between the catalytic residues and the bound nucleoside phosphate implying that 2',5'-linked dinucleoside phosphates may not be the ideal ligands to probe the role of the catalytic amino acid residues. A change in the amino acid sequence in the surface loop region formed by the residues Gly71 to Gly74 drastically affects the conformation of the base binding subsite, and this may account for the inactivity of the enzyme with altered sequence i.e., with Pro, Gly and Ser at positions 71 to 73 respectively. These results thus suggest that in addition to recognition and catalytic sites, interactions at the loop regions which constitute the subsite for base binding are also crucial in determining the substrate specificity.
Balaji
P V
PV
Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
Saenger
W
W
Rao
V S
VS
D001665:Binding Sites; D003198:Computer Simulation; D015226:Dinucleoside Phosphates / Q000737:chemistry / Q000378:metabolism*; D005095:Exoribonucleases / Q000737:chemistry / Q000378:metabolism*; D005998:Glycine / Q000737:chemistry; D006157:Guanosine Monophosphate / Q000378:metabolism*; D006860:Hydrogen Bonding; D008968:Molecular Conformation; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity
D015226:Dinucleoside Phosphates; D012694:Serine; D006157:Guanosine Monophosphate; D005095:Exoribonucleases; C042081:exoribonuclease T; D005998:Glycine