Domain movement in rabbit muscle adenylate kinase might involve proline isomerization

Abstract

The fluorescence probe, 8-anilino-l-naphthalenesulfonic acid (ANS), was used to monitor the induced-fit conformational movement in rabbit muscle adenylate kinase. In 50 mM Tris-HCl buffer (pH 8.1), the time course of ANS binding to rabbit muscle adenylate kinase is a biphasic process. The fast phase completes within the dead-time of the stoppedflow equipment used (about 15 ms), while the slow phase ends in about 10 minutes. In the presence of 2.0 (iM peptidyl prolyl cisl rra/w-isomerase, the rate constant of the slow phase reaction is accelerated about 2.4-fold, suggesting that the domain movement during ANS binding to rabbit muscle adenylate kinase may involve proline isomerization. The activation energy of the slow phase was determined to be 74.6 kJ/mol, which is comparable to the activation energy of proline cis/frans-isomerization (about 80 kJ/mol).

Evidence for at Least Two Native Forms of Rabbit Muscle Adenylate Kinase in Equilibrium in Aqueous Solution

“…The equilibrium constants calculated from both experiments agree very well and the interconversion free energy ⌬G 0 from N 1 to N 2 is calculated according to Equation 31. A previous study reported that the slow phase fluorescence building can be catalyzed by peptidyl prolyl cis/trans isomerases (28). The calculated free energy ⌬G 0 from N 1 to N 2 is close to that of proline isomerization, giving further evidence that the interconversion of the two forms involves the cis/trans peptidyl prolyl isomerization of proline residue.…”

“…The fluorescence intensity of ANS bound to a protein is dependent on the microenvironment of the binding site (32). Experiments wherein AK crystals were soaked with ANS or ATP revealed that ANS or ATP binding occurred only with AK of crystalline form B. ANS occupies the pocket formed between the ␤-sheet, loop 16 -22, helix [23][24][25][26][27][28][29][30], and the C-terminal helix. This pocket was originally assigned to the adenosine moiety of AMP site (12), but has recently been recognized as the ATP site (33,34).…”

“…In a previous study from this laboratory (28), it was found that the time course of 8-anilino-1-naphthalenesulfonic acid (ANS) binding to AK was a biphasic process. The burst phase ended in the dead-time of the stopped-flow apparatus (about 15 ms), whereas the slow phase completed in about 10 min.…”

Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A

“…For the Ca 2ϩ -free form of a C-type mannose binding protein, addition of equimolar amounts of CypA slightly increased the isomerization rate (57). Using 8-anilino-1-naphthalenesulfonic acid (ANS) binding to probe induced-fit conformational changes associated with ATP binding in rabbit muscle adenylate kinase, Sheng et al showed that CypA accelerates ANS binding (58). In both cases, the biological relevance of prolyl isomerization remains unclear.…”

The Influence of Proline Isomerization and Off-Pathway Intermediates on the Folding Mechanism of Eukaryotic UMP/CMP Kinase

“…[11][12][13] Furthermore, proline isomerization very often is observed not only to be responsible for the rate-limiting step in folding but, even more interestingly, also to influence enzymatic activity. 14,15 The link between protein folding and activity makes the 22-kDa uridine monophosphate/cytidine monophosphate (UMP/CMP) kinase from Dictyostelium discoideum (UmpK, 194 amino acids) an interesting model protein to study. UmpK belongs to the family of nucleoside monophosphate (NMP) kinases and can be considered as a relatively large singledomain protein.…”