Dissertation/Thesis Abstract

Investigating the catalytic role of an active site lysine in the ribonuclease a active site
by Chen, Weilee, M.S., California State University, Long Beach, 2016, 82; 10108183
Abstract (Summary)

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.

Indexing (document details)
Advisor: Schwans, Jason
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
Subjects: Chemistry, Biochemistry
Keywords: Lys41, Phosphorothioate, RNase A, Ribonuclease A, UpA
Publication Number: 10108183
ISBN: 978-1-339-71546-9
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