INVESTIGATION OF THE FUNCTIONAL INTERPLAY BETWEEN THE PRIMARY SITE AND THE SUBSITE OF RNASE-T(1) - 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 proper ties of the single Tyr-38-Phe and Asn-98-Ala mutants have been compare d with those of the corresponding double mutant. The Tyr-38-Phe mutati on has been used to probe enzyme-substrate interactions at the primary site; the Asn-98-Ala mutation monitors subsite interactions.1 In addi tion to the dinucleoside phosphate substrate GpC, we measured the kine tics for GpMe, a synthetic substrate in which the leaving nucleoside c ytosine has been replaced by methanol. All data were combined in a tri ple mutant box to analyze the interplay between Tyr-38, Asn-98, and th e leaving group. The free energy barriers to k(cat), introduced by the single Tyr-38-Phe and Asn-98-Ala mutations are not additive in the co rresponding double mutant. The energetic coupling between both mutatio ns is independent of the binding of the leaving cytosine at the subsit e. We conclude that the coupling of the Tyr-38-Phe and Asn-98-Ala muta tions arises through distortion or reorientation of the 3':guanylic ac id moiety bound at the primary site. The experimental data indicate th at the enzyme-substrate interactions beyond the scissile phosphodieste r bond contribute to catalysis through the formation of new or improve d contacts in going from ground state to transition state, which are f unctionally independent of primary site interactions. (C) 1994 Wiley-L iss, Inc.