Both the fast and slow refolding reactions of ribonuclease A yield native enzyme.

The fast reaction ((T2) approximately 50 msec) observed previously in the refolding of thermally unfolded ribonuclease A (disulfide bonds intact) has now been studied by two properties indicative of enzyme function: binding of a competitive inhibitor (2'CMP) and hydrolysis of a substrate (CpA --> C > p + A). Both the binding and catalytic reactions are fast (<2 msec) compared to refolding. Binding of 2'CMP occurs during both fast and slow refolding reactions, and the protein folded in the fast reaction has a normal binding constant for 2'CMP. Recovery of enzymatic activity during the fast refolding reaction, as measured by the rate of CpA hydrolysis, parallels the kinetic curve for 2'CMP binding. When the kinetics of refolding are measured by the burying of exposed tyrosine groups, no difference is found. The presence of 2'CMP has no effect on the kinetics of refolding. The results show that the fast refolding reaction does not yield an intermediate in the refolding of RNase A. Instead, both fast and slow refolding reactions have a common product, fully active RNase A. Although they show a 100-fold difference in rates of refolding, the starting materials for the fast and slow refolding reactions have similar properties, as regards: (a) the molar absorbancy at 286 nm, reflecting the state of exposed tyrosine groups, and (b) their apparent failure to bind 2'CMP.