Both the Fast and Slow Refolding Reactions of Ribonuclease A Yield Native Enzyme

AUTOR(ES)

Garel, Jean-Renaud

RESUMO

The fast reaction (T̄2 ≃ 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.

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