Understanding enzyme catalysis is one of the major goals in biology. Ribonuclease A (RNase A) is a key system to understanding protein structure and function provides an attractive system to investigate the catalytic role of active site interactions. Crystal structures show a lysine residue (Lys41) situated in the RNase A active site, and mutagenesis studies suggest this residue is important for catalysis. To evaluate the catalytic importance of the Lys41-phosphate interaction, double mutant cycle analysis was used. Individual mutation of lysine to arginine (K41R) and substitution of a phosphate oxygen with sulfur led to ∼350 and ∼100-fold decrease in kcat/KM, respectively. However, in the K41R background, substitution of the same oxygen with sulfur decreased activity by a similar amount (within 2-fold) as it did with the wild-type enzyme. This result provides evidence that functional interaction between Lys41 and the phosphate backbone of RNA substrates may not be solely limited between the two groups.
|Advisor:||Schwans, Jason P.|
|Commitee:||Bhandari, Deepali, Weers, Paul M.M.|
|School:||California State University, Long Beach|
|Department:||Chemistry and Biochemistry|
|School Location:||United States -- California|
|Source:||MAI 56/05M(E), Masters Abstracts International|
|Subjects:||Molecular biology, Chemistry, Biochemistry|
|Keywords:||Enzyme catalysis, Enzyme kinetics, Enzymology, Phosphorothioate, Ribonuclease a, Site-directed mutagenesis|
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