Development of enzymatic activity during protein folding - Detection of a spectroscopically silent native-like intermediate of muscle acylphosphatase

The recovery of enzymatic activity during the folding of muscle acylphospha tase and two single residue mutants (proline 54 to alanine and proline 71 t o alanine) from 7 M urea has been monitored and compared with the developme nt of intrinsic fluorescence emission. Fluorescence measurements reveal the presence in the wild-type protein of a major rapid refolding phase followe d by a second low amplitude slow phase. The slow phase is absent in the flu orescence trace acquired with the proline 54 to alanine mutant, suggesting the involvement of this proline residue in the fluorescence-detected slow p hase of the wild-type protein. The major kinetic phase is associated with a considerable recovery of enzymatic activity, indicating that a large fract ion of molecules refolds with effective two-state behavior. The use of time -resolved enzymatic activity as a probe to follow the folding process revea ls, however, the presence of another exponential slow phase arising from pr oline 71. This slow phase is not observable by utilizing optical probes, in dicating that, unlike proline 54, the cis to trans isomerization of proline 71 can take place in an intermediate possessing a native-like fold. We sug gest that, although spectroscopically silent and structurally insignificant , the cia-trans interconversion of proline residues in native-like intermed iates may be crucial for the generation of enzymatic activity of functional enzymes.