Evidence for flexibility in the function of ribonuclease A

Abstract

The dynamic properties of the enzyme ribonuclease A (RNase A) were investigated through the use of solution NMR spin relaxation experiments. As determined by "model-free" analysis, RNase A is conformationally rigid on time scales faster than overall rotational tumbling (picoseconds to nanoseconds). The average order parameter, S(2), for RNase A is 0.910 +/- 0.051. However, 28 of the amino acid residues in RNase A were identified as undergoing chemical exchange on the microsecond to millisecond time scale. For 16 of these residues the microscopic chemical exchange rates, k(ex), were quantitated through the use of the relaxation-compensated CPMG (rcCPMG) experiment. The value of k(ex) was identical for all residues with an average of 1640 s(-1) and is similar to the RNase A k(cat) value of 1900 s(-1). Many of these mobile residues localize to the active site in RNase A and include the catalytically crucial amino acids His119 and Asp121. Additional motion is found in the B1, B2, and P0 subsites, suggesting a coupling of motion between the binding and catalytic sites. The activation energy of the observed millisecond motion was measured by applying the rcCPMG experiment at temperatures of 283, 293, and 298 K and was determined to vary between 3.6 and 7.4 kcal/mol. The measured barrier to conformational motion is similar to the activation barrier for the RNase A catalyzed reaction and thus would not be thermodynamically limiting to catalysis. These studies suggest a correlation of conformational exchange kinetics and thermodynamics derived from NMR measurements with those determined by biochemical means and are suggestive of an important role for flexibility in enzyme function.