Ribonuclease A: Exploring the Function of the Active-Site Lysine Residue in Catalysis and Inhibition

June M. Messmore Under the supervision of Professor Ronald T. Raines at the University of Wisconsin-Madison 1 Structural analyses had suggested that the active-site lysine residue of RNase A (Lys41) may interact preferentially with the transition state for covalent bond cleavage, thus facilitating catalysis. Site-directed mutagenesis and chemical modification were combined (1) to probe the role of Lys41 in catalysis, (2) to provide a chemical on/off switch for RNase A activity in in vitro applications, and (3) to probe the analogy ofuridine 2',3'-cyclic vanadate to the catalytic transition state. In addition to studying inhibition by the uridine vanadate, inhibition by a polyvanadate species was also characterized. Results indicate the importance of positive charge and the donation of a single hydrogen bond in catalysis. Chemical modification can reversibly modulate the activity of ribonuclease by a factor of 30,000. A polyvanadate species, apparently decavanadate, was observed to be a hyperbolically competitive inhibitor of RNase A whose binding is sensitive to ionic strength. Lastly, studies of uridine 2',3'-cyclic vanadate, using RNase A variants altered at position 41, showed that any interaction between this vanadate species and Lys41 is not highly analogous to the interaction between the transition state and Lys41.