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Cis‐trans isomerization is rate‐determining in the reactivation of denatured human carbonic anhydrase II as evidenced by proline isomerase
Author(s) -
Fransson Cecilia,
Freskgård Per-Ola,
Herbertsson Helena,
Johansson Åsa,
Jonasson Per,
Mårtensson Lars-Göran,
Svensson Magdalena,
Jonsson Bengt-Harald,
Carlsson Uno
Publication year - 1992
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(92)80410-i
Subject(s) - isomerase , chemistry , carbonic anhydrase , isomerization , asparagine , proline , mutant , enzyme , carbonic anhydrase ii , biochemistry , stereochemistry , kinetics , amino acid , catalysis , gene , physics , quantum mechanics
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 Pro 30 and Pro 202 . 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 Pro 202 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.