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PubMed2002

Solution structure of the single-domain prolyl cis/trans isomerase PIN1At from Arabidopsis thaliana.

The 119-amino acid residue prolyl cis/trans isomerase from Arabidopsis thaliana (PIN1At) is similar to the catalytic domain of the human hPIN1. However, PIN1At lacks the N-terminal WW domain that appears to be essential for the hPIN1 function. Here, the solution structure of PIN1At was determined by three-dimensional nuclear magnetic resonance spectroscopy. The PIN1At fold could be superimposed on that of the catalytic domain of hPIN1 and had a 19 residue flexible loop located between strand beta1 and helix alpha1. The dynamical features of this beta1/alpha1-loop, which are characteristic for a region involved in protein-protein interactions, led to exchange broadening in the NMR spectra. When sodium sulfate salt was added to the protein sample, the beta1/alpha1 loop was stabilized and, hence, a complete backbone resonance assignment was obtained. Previously, with a phospho-Cdc25 peptide as substrate, PIN1At had been shown to catalyze the phosphoserine/phosphothreonine prolyl cis/trans isomerization specifically. To map the catalytic site of PIN1At, the phospho-Cdc25 peptide or sodium sulfate salt was added in excess to the protein and chemical shift changes in the backbone amide protons were monitored in the (1)H(N)-(15)N heteronuclear single quantum coherence spectrum. The peptide caused perturbations in the loops between helix alpha4 and strand beta3, between strands beta3 and beta4, in the alpha3 helix, and in the beta1/alpha1 loop. The amide groups of the residues Arg21 and Arg22 showed large chemical shift perturbations upon phospho-Cdc25 peptide or sulfate addition. We conclude that this basic cluster formed by Arg21 and Arg22, both located in the beta1/alpha1 loop, is homologous to that found in the hPIN1 crystal structure (Arg68 and Arg69), which also is involved in sulfate ion binding. We showed that the sulfate group competed for the interaction between PIN1At and the phospho-Cdc25 peptide. In the absence of the WW domain, three hydrophobic residues (Ile33, Ile34, and Leu35) located in the long flexible loop and specific for the plant PIN-type peptidyl prolyl cis/trans isomerases (PPIases) could be an additional interaction site in PIN1At. However, phospho-peptide addition did not affect the resonances of these residues significantly. Electrostatic potential calculations revealed a negatively charged area not found in hPIN1 on the PIN1At molecular surface, which corresponds to the surface shielded by the WW domain in hPIN1. Based on our experimental results and the molecular specificities of the PIN1At enzyme, functional implications of the lack of WW domains in this plant PIN-type PPIase will be discussed.

Amino Acid Sequence↗

PubMed1995

Catalytic mechanism of DsbA and its comparison with that of protein disulfide isomerase.

The mechanism of action of the bacterial periplasm protein DsbA in introducing disulfide bonds into proteins was studied by its action on a model disordered peptide containing only two cysteine residues. Most of the reactions between the various thiol and disulfide forms of the peptide and of DsbA could be measured directly. All those involving DsbA occurred 10(2)-10(6) times more rapidly than is normally observed between other typical thiols and disulfides; DsbA apparently stabilizes the transition state of thiol-disulfide exchange. The reactions between DsbA and the peptide were even more rapid, and they were constrained to occur at only one sulfur atom of disulfide bonds involving the peptide. Both observations indicate that noncovalent binding interactions also occur between DsbA and the peptide, and the expected effect of binding between reactants on rates of reaction was quantified. Small quantities of DsbA had catalytic effects on the reaction between the peptide and glutathione, similar to those observed previously with the eukaryotic catalyst protein disulfide isomerase. The known reactions of DsbA could account quantitatively for these effects and indicated that the apparent catalysis was the result of the separate and sequential rapid reactions of the peptide and of glutathione at the active site of DsbA. DsbA did not catalyze the conformational changes involved in forming an intramolecular disulfide bond in the peptide; its catalytic effects were simply due to its rapid participation in thiol-disulfide exchange reactions. Protein disulfide isomerase is likely to function very similarly to DsbA.

Amino Acid Sequence↗

PubMed1992

Conserved residues flanking the thiol/disulfide centers of protein disulfide isomerase are not essential for catalysis of thiol/disulfide exchange.

Protein disulfide isomerase (PDI) catalyzes the oxidative folding of proteins containing disulfide bonds by increasing the rate of disulfide bond rearrangements which normally occur during the folding process. The amino acid sequences of the N- and C-terminal redox active sites (PWCGHCK) in PDI are completely conserved from yeast to man and display considerable identity with the redox-active center of thioredoxin (EWCGPCK). Available data indicate that the two thiol/disulfide centers of PDI can function independently in the isomerase reaction and that the cysteine residues in each active site are essential for catalysis. To evaluate the role of residues flanking the active-site cysteines of PDI in function, a variety of mutations were introduced into the N-terminal active site of PDI within the context of both a functional C-terminal active site and an inactive C-terminal active site in which serine residues replaced C379 and C382. Replacement of non-cysteine residues (W34 to Ser, G36 to Ala, and K39 to Arg) resulted in only a modest reduction in catalytic activity in both the oxidative refolding of RNase A and the reduction of insulin (10-27%), independent of the status of the C-terminal active site. A somewhat larger effect was observed with the H37P mutation where approximately 80% of the activity attributable to the N-terminal domain (approximately 40%) was lost. However, the H37P mutant N-terminal site expressed within the context of an inactive C-terminal domain exhibits 30% activity, approximately 70% of the activity of the N-terminal site alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Characterization of folding intermediates using prolyl isomerase.

Structure-reactivity relationships of human peptidyl prolyl cis-trans isomerase (PPI) toward the two slow folding reactions of yeast iso-2 cytochrome c have been used to characterize the structure of folding intermediates in the vicinity of critical prolines. We propose that the relative catalytic efficiency of PPI for the protein substrate relative to a peptide substrate, (kcat/Km)rel, is a measure of structure in folding intermediates. The structural stability of slow-folding intermediates as detected by changes in (kcat/Km)rel was investigated using two structural perturbants: guanidine hydrochloride and site-directed mutagenesis. Neither of the two slow folding reactions for wild-type cytochrome c is catalyzed at low denaturant concentrations. However, both phases are catalyzed at moderate concentrations of guanidine hydrochloride. A mutation in cytochrome c enhances catalysis of the fluorescence-detected slow folding phase. For protein substrates destabilized by denaturants or mutation, we suggest that increases in (kcat/Km)rel result from a loosening of the substrate structure, providing better access of peptidyl prolyl isomerase to critical proline(s).

Amino Acid Isomerases↗

PubMed1994

Solution conformation of a cyclophilin-bound proline isomerase substrate.

Cyclophilin (CyP) is the 17.8-kDa cytosolic receptor of the immunosuppressant cyclosporin A (CsA) and also a peptidyl prolyl cis-trans isomerase (PPIase). In order to gain insights into the PPIase mechanism, transferred nuclear Overhauser effect (TRNOE) measurements by two-dimensional 1H NMR were used to determine the conformation of the isomerase-bound standard model substrate suc-AAPF-pNA. Results indicate a cis-like conformation for the CyP-bound substrate with the A-P peptide bond being no more than 40 degrees out of planarity.

Amino Acid Isomerases↗

PubMed1994

Identification of Cys139 and Glu207 as catalytically important groups in the active site of isopentenyl diphosphate:dimethylallyl diphosphate isomerase.

Isopentenyl diphosphate:dimethylallyl diphosphate isomerase (EC 5.3.3.2) catalyzes the antarafacial [1.3] allylic rearrangement of isopentenyl diphosphate (IPP) to its electrophilic allylic isomer dimethylallyl diphosphate (DMAPP). Active-site thiols at C138 and C139 were recently identified by covalent modification using active-site-directed irreversible inhibitors [Street, I. P., & Poulter, C. D. (1990) Biochemistry 29, 7531-7538; Lu, X. J., Christensen, D. J., & Poulter, C. D. (1992) Biochemistry 31, 9955-9960]. Kinetic studies were conducted with site-directed mutants of IPP isomerase (IPPIase) to evaluate the roles of these amino acids. C138S and C138V mutants were active catalysts with V/K values only 10-fold lower than that of wild-type IPPIase. In contrast, the C139S mutant was a poor catalyst, and the C139A and C139V mutants were inactive. Treatment of the C139S mutant with 3-(fluoromethyl)-3-butenyl diphosphate, an electrophilic active-site-directed irreversible inhibitor, resulted in inactivation of the enzyme by covalent modification of E207. The E207Q and E207V mutants were inactive, suggesting a role for the E207 carboxylate moiety in catalysis.

Amino Acid Sequence↗

PubMed1994

Protein folding activities of Escherichia coli protein disulfide isomerase.

DsbA is an Escherichia coli periplasmic protein that mediates disulfide bond formation in newly secreted proteins in vivo. Addition of thiol reagents to purified dsbA reduces its disulfide bond and yields disulfide isomerase activity after removal of the thiol reagent. DsbA can catalyze the conversion of a stable misfolded protein, misfolded IGF-I (mis-IGF-I), to its correctly folded conformation under physiological conditions. This conversion is the result of breaking and re-forming two disulfide bonds. The uncatalyzed rate of this reaction is undetectable. Kinetic analysis of the reaction yielded a Km of 43 microM and a kcat of 0.2 min-1. The oxidized form of dsbA stimulates the oxidative folding of completely reduced IGF-I at pH 7.0. Thus, dsbA has two possible functions depending on its redox state. The reduced form of the protein is a disulfide isomerase while the oxidized protein can assist formation of disulfide bonds in reduced substrates under physiological conditions.

Amino Acid Sequence↗

PubMed2001

Folding mechanism of ketosteroid isomerase from Comamonas testosteroni.

Ketosteroid isomerase (KSI) from Comamonas testosteroni is a homodimeric enzyme with 125 amino acids in each monomer catalyzing the allylic isomerization reaction at rates comparable to the diffusion limit. Kinetic analysis of KSI refolding has been carried out to understand its folding mechanism. The refolding process as monitored by fluorescence change revealed that the process consists of three steps with a unimolecular fast, a bimolecular intermediate, and most likely unimolecular slow phases. The fast refolding step might involve the formation of structured monomers with hydrophobic surfaces that seem to have a high binding capacity for the amphipathic dye 8-anilino-1-naphthalenesulfonate. During the refolding process, KSI also generated a state that can bind equilenin, a reaction intermediate analogue, at a very early stage. These observations suggest that the KSI folding might be driven by the formation of the apolar active-site cavity while exposing hydrophobic surfaces. Since the monomeric folding intermediate may contain more than 83% of the native secondary structures as revealed previously, it is nativelike taking on most of the properties of the native protein. Urea-dependence analysis of refolding revealed the existence of folding intermediates for both the intermediate and slow steps. These steps were accelerated by cyclophilin A, a prolyl isomerase, suggesting the involvement of a cis-trans isomerization as a rate-limiting step. Taken together, we suggest that KSI folds into a monomeric intermediate, which has nativelike secondary structure, an apolar active site, and exposed hydrophobic surface, followed by dimerization and prolyl isomerizations to complete the folding.

Aldose-Ketose Isomerases↗

PubMed1991

Two distinct forms of peptidylprolyl-cis-trans-isomerase are expressed separately in periplasmic and cytoplasmic compartments of Escherichia coli cells.

Peptidylprolyl-cis-trans-isomerase (PPIase) is thought to be essential for protein folding in the cell. Two forms, a and b, of PPIase and their corresponding genes were isolated from Escherichia coli cells. Despite their insensitivity to cyclosporin A (CsA), both amino acid sequences were homologous and related to that of pig cyclophilin, a protein that has PPIase activity sensitive to CsA (Takahashi et al., 1989). PPIase a is found to be identical with the E. coli ORF 190 gene product that was sequenced by Kawamukai et al. (1989) and overexpressed by Liu and Walsh (1990). It is translocated into E. coli periplasmic space with the signal sequence. PPIase b lacks a hydrophobic amino acid stretch which could serve as a signal sequence or a transmembrane domain, and it is detected mainly in the bacterial cytoplasm. These findings indicate that proteins with the ability to assist folding of various polypeptides are located on both sides of the inner membrane. Thus, we propose that the folding of some exported proteins may be catalyzed by the periplasmic proline isomerase and, in turn, that some proteins which have isomerized may not be translocated efficiently.

Amino Acid Isomerases↗

PubMed1985

Use of a solid-phase photoaffinity reagent to label a steroid binding site: application to the delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni.

In order to extend our analysis of the reactions that occur during the active site directed photoinactivation of delta 5-3-ketosteroid isomerase sensitized by unsaturated steroid ketone photoaffinity reagents, the site of covalent attachment has been identified. A solid-phase photoaffinity reagent, delta 6-testosterone-agarose, has been employed for this purpose; this type of reagent, in contrast to solution-phase reagents, facilitated the recovery of a peptide fragment of the isomerase bearing the residue at which covalent attachment had occurred. Amino acid analysis and sequence determination of the peptide provided evidence that the site of attachment was aspartate-38. This result, in combination with the low-resolution crystallographic structure of the enzyme [Westbrook, E. M., Piro, O. E., & Sigler, P. B. (1984) J. Biol. Chem. 259, 9096-9103], suggests that aspartate-38 is located in the vicinity of the bottom of the steroid-binding pit. The potential usefulness of solid-phase photoaffinity reagents in the identification of sites of covalent attachment on target proteins such as hormone receptors is discussed.

Affinity Labels↗

PubMed1986

pH dependence of the kinetic parameters for 3-oxo-delta 5-steroid isomerase. Substrate catalysis and inhibition by (3S)-spiro[5 alpha-androstane-3,2'-oxiran]-17-one.

The pH-rate profiles for kcatobsd and (kcat/KM)obsd at 25.0 degrees C have been measured for 3-oxo-delta 5-steroid isomerase by using 5-androstene-3,17-dione (2), 5-pregnene-3,20-dione (3), and 5(10)-estrene-3,17-dione (4) as substrates. Results from the nonsticky substrate 4 suggest values for the pK of a catalytically important group on the free enzyme (pKE) of 4.57 and the pK of the same group in the enzyme-substrate complex of 4.74. For the sticky substrates 2 and 3, pKES is ca. 4.75 and 5.5, respectively. Analysis of the (kcat/KM)obsd vs. pH profile for 2 reveals that the intermediate E X S complex decomposes to products at a rate similar to its reversion to E + S. The pH-rate profile for inhibition of the isomerase by (3S)-spiro-[5 alpha-androstane-3,2'-oxiran]-17-one (7 beta) shows values for pKE of 4.75 and pKEI of 4.90. The similarity of the pH-rate profiles for isomerization of 4 and inhibition by 7 beta suggests that both reactions may be governed by the ionization state of the same carboxyl group of the enzyme.

Androstanes↗

PubMed1987

Affinity alkylation of 3-oxo-delta 5-steroid isomerase by steroidal 3 beta-oxiranes: identification of the modified amino acid by reduction with hydroxyborohydride.

The steroidal 3 beta-oxirane (3S)-spiro[5 alpha-androstane-3,2'-oxiran]-17 beta-ol (1 beta) is an active site directed irreversible inhibitor of the 3-oxo-delta 5-steroid isomerase from Pseudomonas testosteroni. Two steroid-bound peptides (TPS1 and TPS2) were isolated by high-performance liquid chromatography (HPLC) from the trypsin digest of enzyme inactivated with 1 beta. The modified tryptic peptides (residues 14-45 of the enzyme) were further digested with chymotrypsin, each giving rise to a single steroid-containing product (CPS1 and CPS2, respectively) derived from residues 31 to 45 of the enzyme. The modified chymotryptic peptides were isolated by HPLC, and the peptide-steroid ester linkage was reduced with sodium hydroxyborohydride. Amino acid analysis of the reduced peptides gave ca. 0.5 residue of homoserine and one less residue of aspartic acid than the corresponding unreduced peptides. Sequence analysis of both reduced chymotryptic peptides revealed that homoserine was located at position 8 in the peptide sequence, corresponding to residue 38 of the enzyme. The finding that the steroidal 3 beta-oxirane, like the 17 beta-oxiranes, inactivates the isomerase via esterification of aspartic acid-38 is strong evidence that this enzyme binds steroids in at least two orientations.

Amino Acid Sequence↗

PubMed1989

3 Beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase from bovine adrenals: mechanism of inhibition by 3-oxo-4-aza steroids and kinetic mechanism of the dehydrogenase.

Several 3-oxo-4-aza steroids (1) have been identified as inhibitors of the 3 beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase catalyzed conversion of pregnenolone to progesterone. By kinetically decoupling the two enzyme activities isolated from bovine adrenal cortex, it has been demonstrated that inhibition by 1 occurs through interference of both activities. A preferred ordered association of substrates to the 3 beta-hydroxy-delta 5-steroid dehydrogenase in which the cofactor binds prior to steroid was determined by isotope exchange at equilibrium. With this result, the dead-end inhibition patterns of 1 with the dehydrogenase were interpreted to originate from a preferred association of inhibitor within an enzyme ternate containing NADH; this proposal is supported by data from multiple inhibition analysis indicating synergistic binding of NADH and 1. Similarly, inhibition of the 3-keto-delta 5-steroid isomerase by the 3-oxo-4-aza steroids was enhanced in the presence of the positive effector NADH. On the basis of pH profiles upon Vm, Vm/Km, and 1/Ki for both enzyme activities, inhibition is proposed to result from the structural similarity of 1 to intermediate states formed upon enzyme catalysis.

3-Hydroxysteroid Dehydrogenases↗

PubMed1989

Orientation, accessibility, and mobility of equilenin bound to the active site of steroid isomerase.

The fluorescent aromatic steroid equilenin, which contains a beta-naphthol moiety, is bound by 3-oxo-delta 5-steroid isomerase. The excitation and emission fluorescence spectra of equilenin when bound to the enzyme, as well as the fluorescence decay time, are indicative of ground-state ionization. In view of the high efficiency of tyrosine quenching, which approaches 100%, the beta-naphthol moiety of equilenin must be in proximity to all three tyrosines of steroid isomerase to account for the observed efficiency of radiationless energy transfer. From the observed response to an external quencher, it appears that enzyme-bound equilenin is largely shielded from solvent. Fluorescence anisotropy measurements indicate a high degree of immobilization of the bound ligand. These models are consistent with proposed models of the enzyme-substrate complex.

17-Ketosteroids↗

PubMed1990

Mechanistic studies of peptidyl prolyl cis-trans isomerase: evidence for catalysis by distortion.

Cyclophilin, the cytosolic binding protein for the immunosuppressive drug cyclosporin A, has recently been shown to be identical with peptidyl prolyl cis-trans isomerase [Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T., & Schmid, F.X. (1989) Nature 337, 476; Takahashi, N., Hayano, T., & Suzuki, M. (1989) Nature 337, 473]. To provide a mechanistic framework for studies of the interaction of cyclophilin with cyclosporin, we investigated the mechanism of the PPI-catalyzed cis to trans isomerization of Suc-Ala-Xaa-cis-Pro-Phe-pNA (Xaa = Ala, Gly). Our mechanistic studies of peptidyl prolyl cis-trans isomerase include the determination of steady-state kinetic parameters, pH and temperature dependencies, and solvent and secondary deuterium isotope effects. The results of these experiments support a mechanism involving catalysis by distortion in which the enzyme uses free energy released from favorable, noncovalent interactions with the substrate to stabilize a transition state that is characterized by partial rotation about the C-N amide bond.

Amino Acid Isomerases↗

PubMed1990

Substrate specificities of the peptidyl prolyl cis-trans isomerase activities of cyclophilin and FK-506 binding protein: evidence for the existence of a family of distinct enzymes.

Substrate specificities, as reflected in kc/Km, were determined for the peptidyl prolyl cis-trans isomerase activities of cyclophilin and the FK-506 binding protein (FKBP). The substrates investigated were peptides of the general structure Suc-Ala-Xaa-Pro-Phe-p-nitroanilide, where Xaa = Gly, Ala, Val, Leu, Phe, His, Lys, on Glu. While kc/Km for cyclophilin-catalyzed isomerization shows little dependence on Xaa, kc/Km values for FKBP-catalyzed isomerization display a marked dependence on Xaa and vary over 3 orders of magnitude. An important outcome of this work is the discovery that Suc-Ala-Leu-Pro-Phe-pNA is a reactive substrate for FKBP (kc/Km = 640,000 M-1 s-1). This substrate can be used with FKBP concentrations that are low enough to allow, for the first time, accurate determinations of Ki values for tight-binding inhibitors of FKBP. Using this new assay, we found that FK-506 inhibits FKBP with Ki = 1.7 +/- 0.6 nM. The results of this work support the hypothesis that cyclophilin and FKBP are members of a family of peptidyl prolyl cis-trans isomerases and that the members of this family possess distinct substrate specificities that allow them to play diverse physiologic roles.

Amino Acid Isomerases↗

PubMed1996

Catalysis of oxidative protein folding by mutants of protein disulfide isomerase with a single active-site cysteine.

Protein disulfide isomerase (PDI), a very abundant protein in the endoplasmic reticulum, facilitates the formation and rearrangement of disulfide bonds using two nonequivalent redox active-sites, located in two different thioredoxin homology domains [Lyles, M. M., & Gilbert, H. F. (1994) J. Biol. Chem. 269, 30946-30952]. Each dithiol/disulfide active-site contains the thioredoxin consensus sequence CXXC. Four mutants of protein disulfide isomerase were constructed that have only a single active-site cysteine. Kinetic analysis of these mutants show that the first (more N-terminal) cysteine in either active site is essential for catalysis of oxidation and rearrangement during the refolding of reduced bovine pancreatic ribonuclease A (RNase). Mutant active sites with the sequence SGHC show no detectable activity for disulfide formation or rearrangement, even at concentrations of 25 microM. The second (more C-terminal) cysteine is not essential for catalysis of RNase disulfide rearrangements, but it is essential for catalysis of RNase oxidation, even in the presence of a glutathione redox buffer. Mutant active sites with the sequence CGHS show 12%-50% of the kcat activity of wild-type active sites during the rearrangement phase of RNase refolding but < 5% activity during the oxidation phase. In addition, mutants with the sequence CGHS accumulate significant levels of a covalent PDI-RNase complex during steady-state turnover while the wild-type enzyme and mutants with the sequence SGHC do not. Since both active-site cysteines are essential for catalysis of disulfide formation, the dominant mechanism for RNase oxidation may involve direct oxidation by the active-site PDI disulfide. Although it is not essential for catalysis of RNase rearrangements, the more C-terminal cysteine does contribute 2-8-fold to the rearrangement activity. A mechanism for substrate rearrangement is suggested in which the second active-site cysteine provides PDI with a way to "escape" from covalent intermediates that do not rearrange in a timely fashion. The second active-site cysteine may normally serve the wild-type enzyme as an internal clock that limits the time allowed for intramolecular substrate rearrangements.

Amino Acid Sequence↗

PubMedDate not supplied

Catalysis of protein folding by prolyl isomerase.

Rates of protein folding reactions vary considerably. Some denatured proteins regain the native conformation within milliseconds or seconds, whereas others refold very slowly in the time range of minutes or hours. Varying folding rates are observed not only for different proteins, but can also be detected for single polypeptide species. This originates from the co-existence of fast- and slow-folding forms of the unfolded protein, which regain the native state with different rates. The proline hypothesis provides a plausible explanation for this heterogeneity. It assumes that the slow-folding molecules possess non-native isomers of peptide bonds between proline and another residue, and that crucial steps in the refolding of the slow-folding molecules are limited in rate by the slow reisomerization of such incorrect proline peptide bonds. Recently the enzyme peptidyl-prolyl cis-trans isomerase (PPIase) was discovered and purified from pig kidney. It catalyses efficiently the cis in equilibrium trans isomerization of proline imidic peptide bonds in oligopeptides. Here we show that it also catalyses slow steps in the refolding of a number of proteins of which fast- and slow-folding species have been observed and where it was suggested that proline isomerization was involved in slow refolding. The efficiency of catalysis depends on the accessibility for the isomerase of the particular proline peptide bonds in the refolding protein chain.

Amino Acid Isomerases↗