While ribonuclease A is a well studied enzyme, the catalytic role of an active site lysine (Lys41) is not understood, as Lys41 was suggested to interact with the scissile phosphate of the substrate and/or interact with the 2’-hydroxyl nucleophile. To distinguish these models, we sought to use a double mutant cycle analysis. Lys41 was mutated to alanine and arginine, and the phosphate oxygens were individually substituted with sulfur. Challenges were encountered including obtaining sufficient quantities of pure enzyme and reproducible kinetics results. Nevertheless, our results show the Sp phosphorothioate decreased activity ∼10-fold, while the Rp substitution led to a ∼300-fold decrease. Structures show the pro-Rp oxygen situated to interact with Lys41, and an important role for this interaction in catalysis is supported by the larger deleterious effect from substitution at the Rp site. The results provide a foundation for using double mutant cycle analysis to investigate ribonuclease A catalysis.
|Commitee:||McAbee, Douglas, Weers, Paul|
|School:||California State University, Long Beach|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- California|
|Source:||MAI 55/04M(E), Masters Abstracts International|
|Keywords:||Lys41, Phosphorothioate, RNase A, Ribonuclease A, UpA|
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