Trp59 to Tyr substitution enhances the catalytic activity of RNase T1 and of the Tyr to Trp variants in positions 24, 42 and 45.
Using point mutated overproducing strains of E. coli, ribonuclease T1 was prepared with the single substitutions Tyr24Trp, Tyr42Trp, Tyr45Trp or Trp59Tyr and the corresponding double substitutions Tyr24Trp/Trp59Tyr, Tyr42Trp/Trp59Tyr and Tyr45Trp/Trp59Tyr. Steady state kinetics of the transesterification reaction for the two dinucleoside monophosphate substrates guanylyl-3',5'-cytidine and guanylyl-3',5'-adenosine indicate that the tryptophan can be introduced in different positions within the ribonuclease T1 molecule without abolishing enzymatic activity. The Trp59Tyr exchange even enhances catalysis of the cleavage reaction (kcat/Km) relative to the wild type enzyme and similar effects are found with single tyrosine to tryptophan substitutions. For the pH dependencies of the guanylyl-3',5'-cytidine transesterification reaction of wild type ribonuclease T1 and of the variants, typically bell-shaped curves are observed with a plateau in the range pH 4.5-7.0. Their shapes and slopes indicate that the enzymes are comparable in their macroscopic pKa values. At pH 7.5, the variant Tyr45Trp/Trp59Tyr shows a more than 3-fold higher transesterification activity for guanylyl-3',5'-adenosine and a 2-fold increase for guanylyl-3',5'-cytidine compared to the wild type enzyme, i.e. this variant catalyses the transesterification of the substrate guanylyl-3',5'-adenosine with the same or better efficiency as guanylyl-3',5'-cytidine.
Grunert
H P
HP
Institut für Biochemie, Medizinische Universität zu Lübeck, Germany.
Landt
O
O
Zirpel-Giesebrecht
M
M
Backmann
J
J
Heinemann
U
U
Saenger
W
W
Hahn
U
U
D001483:Base Sequence; D002384:Catalysis; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004951:Esterification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000235:genetics; D014364:Tryptophan; D014443:Tyrosine
D015226:Dinucleoside Phosphates; D014443:Tyrosine; C004804:guanylyl-(3',5')-cytidine; C013011:guanine ribonucleotidyl-(3'-5')-adenosine; D014364:Tryptophan; D006163:Ribonuclease T1