Functional interactions among the His40, Glu58 and His92 catalysts of ribonuclease T1 as studied by double and triple mutants.
We report on the functional interplay between the His40, Glu58 and His92 catalysts of ribonuclease T1. The kinetic properties of the single His40Ala, Glu58Ala and His92Gln mutants have been compared with those of the corresponding double and triple mutants. When His40, Glu58 and His92 are mutated separately or together, we observe large effects on turnover but only minor effects on substrate binding. The free energy barriers to kcat introduced by the single His40Ala, Glu58Ala and His92Gln mutations are non-additive in the corresponding His40Ala + Glu58Ala, Glu58Ala + His92Gln and His40Ala + His92Gln double mutants; a significant dependence of the pairwise interactions on the third residue has been observed. Using a pair of related triple mutant boxes, we were able to divide the apparent coupling energies as calculated from the kinetic parameters of the various mutants either quantitatively or qualitatively into terms that measure intrinsic interaction energies between His40, Glu58 and His92 in wild-type enzyme and terms that account for a change in reaction mechanism that is associated with the Glu58Ala single mutation. In most cases, non-additivity may be explained by changes in enzyme mechanism. Apart from this change in mechanism, functional interactions have to be considered between His40 and Glu58, Glu58 and His92 as well as between His40 and His92 to explain non-additivity in all double-mutant cycles. It is concluded that the collaborative effects of His40, Glu58 and His92 decrease the energetic barrier to kcat by 6.8 kcal/mol. The overall effect caused by the triple mutation is smaller than that expected from the product of the fractional kcat values resulting from the individual mutations (11.0 kcal/mol), illustrating the limitations of using single mutants to probe the energetics of a catalytic group whose function is dependent upon interactions with others.
Steyaert
J
J
Vrije Universiteit Brussel, Instituut Moleculaire Biologie, ULTRASTRUCTUUR, Sint-Genesius-Rode, Belgium.
Wyns
L
L
D002384:Catalysis; D005973:Glutamine / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000235:genetics
D005973:Glutamine; D006639:Histidine; D006163:Ribonuclease T1