Cis‐trans isomerization is rate‐determining in the reactivation of denatured human carbonic anhydrase II as evidenced by proline isomerase

FEBS Letters

Fransson, Cecilia¹  Freskgård, Per-Ola¹  Mårtensson, Lars-Göran²  Svensson, Magdalena¹  Herbertsson, Helena¹  Jonasson, Per²  Johansson, Åsa¹  Carlsson, Uno¹  Jonsson, Bengt-Harald² 

[1] IFM/Department of Chemistry, Linköping University, S-581 83 Linköping, Sweden;Department of Biochemistry, Umeå University, S-901 87 Umeå, Sweden

关键词: cis-trans Isomerization;    Peptidyl proline isomerase;    Carbonic anhydrase;    Refolding;    Reactivation;    Site-directed mutagenesis;    GuHCl;    Guanidine-HCl;    BCA II;    bovine carbonic anhydruse II;    HCA II;    human carbonic anhydrase II;    PPlase;    peptidyl-prolyl cis-trans isomerase;   

DOI  :  10.1016/0014-5793(92)80410-I

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The refolding of human carbonic anhydrase II is a sequential process. The slowest step involved is the recovery of enzymic activity (t_½=9 min). Kinetic data from ‘double-jump’ measurements indicate that proline isomerization might be rate determining, in the reactivation of the denatured enzyme. Proof of this is provided by the effect of proline isomerase on the reactivation kinetics; the presence of isomerase during reactivation lowers the half-time or the reaction to 4 min, and inhibition of proline isomerase completely abolishes this kinetic effect. A similar acceleration of the refolding process by proline isomerase is also observed for bovine carbonic anhydrase II, in contrast to what has previously been reported. In human carbonic anhydrase II there are two cis-peptidyl-Pro bonds at Pro³⁰ and Pro²⁰². Two asparagine single mutants (P30N and P202N) and a glycine double mutant (P30G/P202G) wore constructed to investigate the role of these prolines in the rate limitation of the reactivation process. Both in the presence and absence of PPlase the P202N mutant behaved exactly like the unmutaled enzyme, Thus, cis-trans isomerization of the Pro²⁰² cis-peptidyl bond is not rate determining in the reactivation process, The mutations at position 30 led to such extensive destabilization of the protein that the refolding reaction could not be studied.

【 授权许可】

Unknown