Probing functional perfection in substructures of ribonuclease T-1: Doublecombinatorial random mutagenesis involving Asn43, Asn44, and Glu46 in the guanine binding loop

Combinatorial random mutageneses involving either Asn43 with Asn44 (set 1) or Glu46 with an adjacent insertion (set 2) were undertaken to explore the functional perfection of the guanine recognition loop of ribonuclease T-1 ( RNase T-1), Four hundred unique recombinants were screened in each set for their ability to enhance enzyme catalysis of RNA cleavage. After a thorough selection procedure, only six variants were found that were either as acti ve or more active than wild type which included substitutions of Asn43 by G ly, His, Leu, or Thr, an unplanned Tyr45Ser substitution and Glu46Pro with an adjacent Glu47 insertion. Asn43His-RNase T-1 has the same loop sequence as that for RNases Pb-1 and Fl(2). None of the most active mutants were sin gle substitutions at Asn44 or double substitutions at Asn43 and Asn44, A to tal of 13 variants were purified, and these were subjected to kinetic analy sis using RNA, GpC, and ApC as substrates. Modestly enhanced activities wit h GpC and RNA involved both k(cat) and K-M effects, Mutants having low acti vity with GpC had proportionately even lower relative activity with RNA. As n43Gly-RNase T-1 and all five of the purified mutants in set 2 exhibited si milar values of k(cat)/K-M for ApC which were the highest observed and abou t 1.0-fold that for wild type, The specificity ratio [(k(cat)/ K-M)(GpC)/(k (cat)/K-M)(ApC)] varied over 30 000-fold including a 10-fold increase [Asn4 3His variant; mainly due to a low (k(cat)/K-M)(ApC)] and a 3000-fold decrea se (Glu46Ser/(insert)Gly47 variant; mainly due to a low (k(cat)/K-M)(GpC)) as compared with wild type. It is interesting that k(cat) (GpC) for the Tyr 45Ser variant was almost LC-fold greater than for wild type and that Pro46/ (insert)Glu47 RNase T-1 is 70-fold more active than the permuted variant (i nsert)Pro47-RNase T-1 which has a conserved Glu46, In any event, the observ ation that only 6 out of 800 variants surveyed had wild-type activity suppo rts the view that functional perfection of the guanine recognition loop of RNase T-1 has been achieved.