Investigation of the functional interplay between the primary site and the subsite of RNase T1: kinetic analysis of single and multiple mutants for modified substrates.
We report on the functional cooperativity of the primary site and the subsite of ribonuclease T1 (RNase T1; EC 3.1.27.3). The kinetic properties of the single Tyr-38-Phe and Asn-98-Ala mutants have been compared with those of the corresponding double mutant. The Tyr-38-Phe mutation has been used to probe enzyme-substrate interactions at the primary site; the Asn-98-Ala mutation monitors subsite interactions. In addition to the dinucleoside phosphate substrate GpC, we measured the kinetics for GpMe, a synthetic substrate in which the leaving nucleoside cytosine has been replaced by methanol. All data were combined in a triple mutant box to analyze the interplay between Tyr-38, Asn-98, and the leaving group. The free energy barriers to kcat, introduced by the single Tyr-38-Phe and Asn-98-Ala mutations are not additive in the corresponding double mutant. The energetic coupling between both mutations is independent of the binding of the leaving cytosine at the subsite. We conclude that the coupling of the Tyr-38-Phe and Asn-98-Ala mutations arises through distortion or reorientation of the 3'-guanylic acid moiety bound at the primary site. The experimental data indicate that the enzyme-substrate interactions beyond the scissile phosphodiester bond contribute to catalysis through the formation of new or improved contacts in going from ground state to transition state, which are functionally independent of primary site interactions.
Steyaert
J
J
Instituut Moleculaire Biologie, Vrije Universiteit Brussel, St.-Genesius-Rode, Belgium.
Haikal
A F
AF
Wyns
L
L
D000596:Amino Acids / Q000235:genetics; D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004252:DNA Mutational Analysis; D008954:Models, Biological; D011994:Recombinant Proteins / Q000378:metabolism; D006163:Ribonuclease T1 / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
D000596:Amino Acids; D011994:Recombinant Proteins; D006163:Ribonuclease T1