Cis-trans isomerization is rate-determining in the reactivation of denatured human carbonic anhydrase II as evidenced by proline isomerase.
The refolding of human carbonic anhydrase II is a sequential process. The slowest step involved is the recovery of enzymic activity (t1/2 = 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 of 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) were constructed to investigate the role of these prolines in the rate limitation of the reactivation process. Both in the presence and absence of PPIase the P202N mutant behaved exactly like the unmutated 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.
Fransson
C
C
IFM/Department of Chemistry, Linköping University, Sweden.
Freskgård
P O
PO
Herbertsson
H
H
Johansson
A
A
Jonasson
P
P
Mårtensson
L G
LG
Svensson
M
M
Jonsson
B H
BH
Carlsson
U
U
D000591:Amino Acid Isomerases / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D019696:Peptidylprolyl Isomerase; D011392:Proline / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation
D002352:Carrier Proteins; D011392:Proline; D002256:Carbonic Anhydrases; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase