title: Intermediates in the refolding of ribonuclease at subzero temperatures. 2. Monitoring by inhibitor binding and catalytic activity.
abstract: The kinetics of refolding of ribonuclease A were monitored by the return of catalytic activity and inhibitor binding at -15 degrees C in 35% methanol cryosolvent at pH* 3.0 and 6.0. Catalytic activity was measured with cytidine 2',3'-cyclic monophosphate as substrate; inhibitor binding was determined with the competitive inhibitor cytidine 2'-monophosphate. Biphasic kinetics were observed at pH* 3.0 for both return of catalytic activity and inhibitor binding. At pH* 6.0 the rate of return of catalytic activity was monophasic, whereas that of inhibitor binding was biphasic. For both inhibitor binding and catalytic activity one of the observed rates was pH-dependent. Full return of catalytic activity was obtained at the completion of the refolding process. The observations are interpreted in terms of two parallel pathways of refolding for slow-refolding ribonuclease, with several native-like, partially folded intermediate states on the minor slow-refolding pathway. Of particular note is the presence of at least one such species that has inhibitor-binding capacity but not catalytic activity. This may be rationalized in terms of the known native structure. In addition, an intermediate is postulated which has the incorrect Pro-93 conformation and only partial catalytic activity (42% of the native). The slowest observed transient is attributed to the isomerization of this proline residue and return of full catalytic activity.
authors.affiliation: Department of Chemistry, University of California, Santa Cruz 95064.
authors.forename: R G
authors.initials: RG
authors.lastname: Biringer
authors.forename: C M
authors.initials: CM
authors.lastname: Austin
authors.forename: A L
authors.initials: AL
authors.lastname: Fink
mesh_terms: D001665:Binding Sites; D003563:Cyclic CMP / Q000378:metabolism; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*
chemical_list: D003563:Cyclic CMP; D012259:Ribonuclease, Pancreatic