Confirmation of the secondary deuterium isotope effect for the peptidyl prolyl cis-trans isomerase activity of cyclophilin by a competitive, double-label technique

Harrison, Richard K.; Caldwell, Charles G.; Rosegay, Avery; Melillo, David G.; Stein, Ross L.

doi:10.1021/ja00175a052

Cited by 30 publications

(21 citation statements)

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“…Subsequently, however, this value was reported as 1.13 AE 0.01 by Harrison and Stein 71 and con®rmed by Harrison et al with a double labeled technique to be 1.11 AE 0.02. 72 These results suggest that the enzyme-catalyzed reaction follows a similar mechanism to that of the uncatalysed reaction with a distorted peptide bond in the transition state (Scheme 2). The difference in isotope effects in the enzymatic and nonenzymatic reactions (1.12±1.05) has been interpreted to suggest a difference in transition state structure with greater hyperconjugation of the b-hydrogens of the transition state in the enzymecatalyzed reaction compared to the non-enzymatic reaction.…”

Section: A Structural Studies Of Immunophilin Proteins Alonementioning

confidence: 75%

Immunophilins: Switched on protein binding domains?

Ivery

2000

Med. Res. Rev.

121

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Peptidylprolyl isomerases (PPIases) are a group of cytosolic enzymes first characterized by their ability to catalyze the cis–trans isomerization of cis‐peptidylprolyl bonds. Subsequently, some PPIases were also identified as the initial targets of the immunosuppressant drugs—cyclosporin A (CsA), FK506, and rapamycin—have been called immunophilins. Immunophilins have been found to be both widely distributed and abundantly expressed leading to suggestions that they may play a general role in cellular biochemistry. However, the nature of this role has been difficult to elucidate and is still controversial in vivo. A number of roles for these enzymes have been identified in vitro including the ability to catalyze the refolding of partly denatured proteins and stabilize multiprotein complexes such as Ca2+ channels, inactive steroid receptor complexes, and receptor protein tyrosine kinases. Generally, these effects appear to depend on the ability of immunophilins to selectively bind to other proteins. This review will examine in detail experimental and structural investigations of the mechanism of PPIase activity for both FKBPs and cyclophilins and suggest a mechanism for these enzymes, which depends on their ability to recognize a specific peptide conformation rather than sequence. Examination of structures of immunophilin–protein complexes will then be used to further suggest that the ability of these enzymes to recognize specific peptide conformations is central to the formation of these complexes and may constitute a general function of immunophilin enzymes. The binding of ligand to immunophilins will also be shown to stabilize specific conformations in surface loops of these proteins that are observed to play a critical role in a number of immunophilin–protein complexes suggesting that the immunophilins may constitute a class of ligand‐triggered selective protein binders. © 2000 John Wiley & Sons, Inc. Med Res Rev, 20, No. 6, 452–484, 2000

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Section: A Structural Studies Of Immunophilin Proteins Alonementioning

confidence: 75%

Immunophilins: Switched on protein binding domains?

Ivery

2000

Med. Res. Rev.

121

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“…The modes of binding of the fungal metabolites and the peptides bearing X-pro epitopes to PPIases have been studied by NMR, X-ray crystallography, isotope effects, theoretical models and synthetic analogues [132][133][134][135][136][137][138][139][140][141][142][143][144][145][146][147][148]. An NMR study on 13 C-enriched FK506 bound to hFKBP-12a and 13 C-CsA bound to hCyP-18a had shown that only the trans form of the immunosuppressive drugs were bound to the respective immunophilins without formation of tetrahedral intermediates [133].…”

Section: ) Models Of Ppiase Activitymentioning

confidence: 99%

“…The macrolides FK506 and rapamycin both have a dicarbonyl moiety whose torsion angle is nearly 90°. This peculiar geometric element of the two immunosuppressive drugs gave rise to suggestion that these drugs inhibit the PPIase activity of immunophilins, since they mimic an intermediate state of the cis/trans peptidylprolyl bond interconversion of peptide and protein substrates: the twisted-amide model [133], or catalysis by distortion [134]. The secondary deuterium effect [135] and analyses of mutants of hFKBP-12a [136] have given some support to the latter model and have not substantiated any mechanism involving nucleophilic catalysis.…”

Section: ) Models Of Ppiase Activitymentioning

confidence: 99%

Peptidylprolyl Cis / Trans Isomerases (Immunophilins): Biological Diversity - Targets - Functions

Gałat

2003

CTMC

342

302

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Information recovered from genome sequencing projects, multiple sequence alignments, structural analyses of PPIase and published records were used in deciphering the biological diversity, functions and targets of four groups of proteins encoded by dissimilar sets of sequences whose spatial representations exhibit peptidylprolyl cis/trans isomerase activity (PPIase). In the human genome there are encoded fifteen proteins whose segments have significant homology with the sequence of 12 kDa protein which is the target of the potent immunosuppressive macrolides FK506 or rapamycin. The 12 kDa archetype of the FK506-binding protein (FKBP), known as FKBP-12a, is an abundant intracellular protein whereas other FKBPs possessing from one to four FK506-like binding domains (FKBDs) have nominal masses varying from 13 to 135 kDa. The human genome contains at least sixteen genes encoding proteins comprising one cyclosporin-A (CsA) binding domain (CLD) called cyclophilins whose nominal masses vary from 17 to 324 kDa and multiple coding segments for small cyclophilins (17-19 kDa) whose transcription levels and functions remain unknown. The third group of PPIases encoded in the genome comprises two proteins (hPin1 and hParv14) where hPin1 is an important PPIase for cell cycle. The A. thaliana, C. elegans, D. melanogaster and S. cerevisiae genomes encode a less diverse spectrum of PPIases whereas the prokaryotic genomes contain from none to three cyclophilins, from none to four genes encoding FKBPs, one distant homologue of the Pin1 protein named parvulin and the fourth group of PPIases known as trigger factors. PPIases are discretely distributed to different cellular compartments and interact with a number of targets that control a range of cellular processes. Analyses of the sequence alignments of the two groups of PPIases, namely cyclophilins and FKBPs from diverse phyla, show that in each group their sequences diverge but the amino acid residues which form the PPIase activity site and macrolide binding cavity remain well conserved in the majority of them which suggests that the spatial structures and functions of each group of PPIases remain conserved.

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“…(10, 11) It has been suggested the PPIases bind the Xaa-Pro substrate and distort the amide bond by pyramidalizing the prolyl nitrogen through hydrogen bonding. (6, 10, 14, 15) Calculation of the FKBP reaction pathway showed substrate intramolecular donation of a hydrogen bond from the C-terminal amide N–H to the prolyl N in the transition state.…”

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confidence: 99%

Kinetic Isotope Effects Support the Twisted Amide Mechanism of Pin1 Peptidyl-Prolyl Isomerase

et al. 2013

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The Pin1 peptidyl-prolyl isomerase (PPIase) catalyzes isomerization of pSer/pThr-Pro motifs in regulating the cell cycle. Peptide substrates, Ac–Phe–Phe–phosphoSer–Pro–Arg–p-nitroaniline, were synthesized in unlabeled form, and with deuterium labeled Ser-d3 and Pro-d7 amino acids. Kinetic data was collected as a function of Pin1 concentration to measure kinetic isotope effects (KIE) on catalytic efficiency (kcat/Km). The normal secondary (2°) KIE value measured for the Ser-d3 substrate (kH/kD = 1.6 ± 0.2) indicates that the serine carbonyl does not rehybridize from sp2 to sp3 in the rate-determining step, ruling out a nucleophilic addition mechanism. The normal 2° KIE can be explained by hyperconjugation between Ser α-C–H/D and C=O, and release of steric strain upon rotation of the amide bond from cis to syn-exo. The inverse 2° KIE value (kH/kD = 0.86 ± 0.08) measured for the Pro-d7 substrate indicates rehybridization of the prolyl nitrogen from sp2 to sp3 during the rate-limiting step of isomerization. No solvent kinetic isotope was measured by NMR exchange spectroscopy (EXSY) (kH2O/kD2O = 0.92 ± 0.12), indicating little or no involvement of exchangeable protons in the mechanism. These results support the formation of a simple twisted-amide transition state as the mechanism for peptidyl prolyl isomerization catalyzed by Pin1. A model of the reaction mechanism is presented using crystal structures of Pin1 with ground state analogues and an inhibitor that resembles a twisted amide transition state.

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Confirmation of the secondary deuterium isotope effect for the peptidyl prolyl cis-trans isomerase activity of cyclophilin by a competitive, double-label technique

Cited by 30 publications

References 0 publications

Immunophilins: Switched on protein binding domains?

Immunophilins: Switched on protein binding domains?

Peptidylprolyl Cis / Trans Isomerases (Immunophilins): Biological Diversity - Targets - Functions

Kinetic Isotope Effects Support the Twisted Amide Mechanism of Pin1 Peptidyl-Prolyl Isomerase

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