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At least 811 records · Page 45Linked to original sources
PubMed1994

Deamidation of triosephosphate isomerase in reverse micelles: effects of water on catalysis and molecular wear and tear.

The specific deamidation of asparagine-71 of triosephosphate isomerase increases upon substrate binding and catalysis. This deamidation at the dimer interface initiates subunit dissociation, unfolding, and protein degradation. The apparent connection between catalysis and terminal marking supports the concept of "molecular wear and tear", and raises questions related to the molecular events that lead to deamidation. In order to explore this interaction, triosephosphate isomerase was entrapped in reverse micelles with different water contents that support different catalytic rates. Deamidation was quantified for the free enzyme, the enzyme in the presence of substrates, and the enzyme which had been covalently modified at the catalytic center with the substrate analogue 3-chloroacetol phosphate (CAP). Both in water and in reverse micelles of cetyltrimethylammonium with 3% and 6% water, substrate binding enhanced deamidation. Studies of the extent of deamidation at various water concentrations showed that deamidation per catalytic turnover was about 6 and 17 times higher in 6% and 3% water than in 100% water, respectively. The enzyme was also entrapped in micelles formed with toluene, phospholipids, and Triton X-100 to explore the process at much lower water concentrations (e.g., 0.3%). Under these conditions, catalysis was very low, and hardly any deamidation took place. Deamidation of the CAP-labeled enzyme was also markedly diminished. At these low-water conditions, the enzyme exhibited markedly increased thermostability and resistance to hydrolysis of the amide bonds. The data suggest that the rate of deamidation not only is dependent on the number of catalytic events but also is related to the time that asparagine-71 exists in a conformation or solvent environment more favorable for deamidation.

Amides↗
PubMed1994

Characterization of the three tyrosine residues of delta 5-3-ketosteroid isomerase by time-resolved fluorescence and circular dichroism.

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni converts delta 5-3-ketosteroids to delta 4-3-ketosteroids via an enolic intermediate. Site-specific mutagenesis has identified Tyr-14 and Asp-38 as the catalytically essential general acid and base, respectively. Three tyrosine residues (Tyr-14, Tyr-55, and Tyr-88) are the only significant fluorophores in the wild-type isomerase. Recent studies of the steady-state fluorescence of the wild-type enzyme and all six mutant enzymes in which one or two tyrosine residues have been mutated to phenylalanine show that the fluorescence intensity of Tyr-14 is very high, that of Tyr-88 is very low, and that of Tyr-55 is intermediate and comparable to that of N-acetyltyrosine amide in solution (Li, Y.-K., Kuliopulos, A., Mildvan, A.S., & Talalay, P. (1993) Biochemistry 32, 1816-1824). Extension of these experiments by time-resolved fluorescence and fluorescence anisotropy measurements demonstrates that Tyr-14, which is in a hydrophobic environment, has an unusually long fluorescence lifetime (4.6 ns) as compared to Tyr-55 (2.0 ns) or Tyr-88 (0.8 ns) and to most protein tyrosine residues (0.2-2 ns). The Förster distances obtained from the absorption and emission of these tyrosines predict that total quenching of Tyr-14 fluorescence by Tyr-55, and to a lesser degree by Tyr-88, would occur if their orientations were favorable.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1993

Identification of Cys-150 in the active site of phosphomannose isomerase from Candida albicans.

Candida albicans phosphomannose isomerase (PMI) (EC 5.3.1.8) has been recently cloned and overexpressed in Escherichia coli. The enzyme can be irreversibly inactivated by iodoacetate in 50 mM borate buffer, pH 9.0, in a time-dependent manner at a rate of 4.2 +/- 0.03 min-1 M-1. This inhibition can be prevented by the substrate mannose 6-phosphate with a Ks of 0.22 +/- 0.05 mM, slightly lower than its Km value. However, metals such as zinc and cadmium, which are reversible, competitive inhibitors for PMI, do not protect the enzyme against modification. The protein has been labeled by using [2-14C]iodoacetate, in the presence or absence of substrate, and the protein is fully inactivated when 1.0 thiol group is modified per molecule of enzyme. Tryptic maps of the modified protein have been produced. The protected peptide has been identified and sequenced, and the phenylthiohydantoin amino acids have been collected. The modified amino acid is Cys-150. This cysteine residue is conserved in mammalian and yeast phosphomannose isomerases, but not in bacterial species where it is replaced with asparagine. We therefore purified PMI from E. coli and showed that this enzyme is not sensitive to inactivation by iodoacetate. The iodoacetate is presumably inhibiting PMI by sterically blocking the mannose 6-phosphate binding site. Multiple sequence alignment procedures were used to try to identify potential ligands of the zinc atom that is essential for enzyme activity and thus to delineate the active site region.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Stereochemistry of the concerted enolization catalyzed by delta 5-3-ketosteroid isomerase.

The reaction catalyzed by delta 5-3-ketosteroid isomerase has been shown to occur via the concerted enolization of the delta 5-3-ketosteroid substrate to form a dienolic intermediate, brought about by Tyr-14, which hydrogen bonds to and protonates the 3-keto group, and Asp-38, which removes and axial (beta) proton from C-4 of the substrate, in the same rate-limiting step [Xue, L., Talalay, P., & Mildvan, A.S. (1990) Biochemistry 29, 7491-7500; Kuliopulos, A., Mildvan, A.S., Shortle, D., & Talalay, P. (1989) Biochemistry 26, 3927-3937]. Since the axial C-4 proton is removed by Asp-38 from above the substrate, a determination of the complete stereochemistry of this rapid, concerted enolization requires information on the direction of approach of Tyr-14 to the enzyme-bound steroid. The double mutant enzyme, Y55F + Y88F, which retains Tyr-14 as the sole Tyr residue, was prepared and showed only a 4.5-fold decrease in kcat (12,000 s-1) and a 3.6-fold decrease in KM (94 microM) for delta 5-androstene-3, 17,dione, in comparison with the wild-type enzyme. Deuteration of the aromatic rings of the 10 Phe residues further facilitated the assignment of the aromatic proton resonances of Tyr-14 in the 600-MHz TOCSY spectrum at 6.66 +/- 0.01 ppm (3,5H) and at 6.82 +/- 0.01 ppm (2,6H). Variation of the pH from 4.9 to 10.9 did not alter these shifts, indicating that the pKa of Tyr-14 exceeds 10.9. Resonances assigned to the three His residues titrated with pKa values very similar to those found with the wild-type enzyme. The binding of 19-nortestosterone, a product analogue and substrate of the reverse isomerase reaction, induced downfield shifts of -0.12 and -0.06 ppm of the 3,5-and 2,6-proton resonances of Tyr-14, respectively, possibly due to deshielding by the 3-keto group of the steroid, but also induced +0.29 to -0.41 ppm changes in the chemical shifts of 8 of the 10 Phe residues and smaller changes in 10 of the 12 ring-shifted methyl resonances, indicating a steroid-induced conformation change in the enzyme. NOESY spectra in H2O revealed strong negative Overhauser effects from the 3,5-proton resonance of Tyr-14 to the overlapping 2 alpha-, 2 beta-, or 6 beta-proton resonances of the bound steroid but no NOE's to the 4- or 6 alpha-protons of the steroid.(ABSTRACT TRUNCATED AT 400 WORDS)

Escherichia coli↗
PubMed1994

Reaction energetics of a mutant 3-oxo-delta 5-steroid isomerase with an altered active site base (D38E).

3-Oxo-delta 5-steroid isomerase (KSI) catalyzes the isomerization of a variety of 3-oxo-delta 5-steroids to their conjugated delta 4-isomers through the formation of an intermediate dienol. Mutation of the catalytic base (Asp-38) to Glu (D38E) has been found to reduce kcat/Km for the isomerization of 5-androstene-3,-17-dione (1) to 4-androstene-3,17-dione (3) by about 300-fold (Zawrotny et al., 1991). The free energy profile for the D38E enzyme was determined from a combination of steady state kinetics and stopped-flow kinetics with the independently generated dienol intermediate (2). A comparison of the energetics of D38E with that of the wild type enzyme (WT) shows that the only significant difference is a reduction in the rates of the chemical steps for the interconversion of 1, 2, and 3 on the enzyme surface by about 10(3)-fold for D38E. The relative energy levels for all bound species are nearly identical for WT and D38E, whereas the transition states for both enolization and ketonization are destabilized by 3-4 kcal/mol. The effect of the D38E mutation on the energetics of KSI is comparable to the corresponding effect of the E165D mutation on the energetics of triosephosphate isomerase (TIM).

Aspartic Acid↗
PubMed1987

Subunit interface of triosephosphate isomerase: site-directed mutagenesis and characterization of the altered enzyme.

We have replaced asparagine residues at the subunit interface of yeast triosephosphate isomerase (TIM) using site-directed mutagenesis in order to elucidate the effects of substitutions on the catalytic activity and conformational stability of the enzyme. The mutant proteins were expressed in a strain of Escherichia coli lacking the bacterial isomerase and purified by ion-exchange and immunoadsorption chromatography. Single replacements of Asn-78 by either Thr or Ile residues had little effect on the enzyme's catalytic efficiency, while the single replacement Asn-78----Asp-78 and the double replacement Asn-14/Asn-78----Thr-14/Ile-78 appreciably lowered kcat for the substrate D-glyceraldehyde 3-phosphate. The isoelectric point of the mutant Asn-78----Asp-78 was equivalent to that of wild-type yeast TIM that had undergone a single, heat-induced deamidation, and this mutant enzyme was less resistant than wild-type TIM to denaturation and inactivation caused by elevated temperature, denaturants, tetrabutylammonium bromide, alkaline pH, and proteases.

Amino Acid Sequence↗
PubMed1987

Enzyme relaxation in the reaction catalyzed by triosephosphate isomerase: detection and kinetic characterization of two unliganded forms of the enzyme.

Triosephosphate isomerase has been shown to exist in two unliganded forms, one of which binds and isomerizes (R)-glyceraldehyde 3-phosphate and the other of which binds and isomerizes dihydroxyacetone 3-phosphate. The tracer perturbation method of Britton demonstrates the kinetic significance of the interconversion of these two enzyme forms at high substrate concentrations and yields a rate constant of about 10(6) s-1 for the interconversion. Although the molecular nature of the two forms of unliganded enzyme is not defined by these experiments, a shuffling of protons among active site residues, or a protein conformational change, or both, may be involved. This study, coupled with the known rate constants for the substrate-handling steps of triosephosphate isomerase catalysis, completes the kinetic characterization of the catalytic cycle for this enzyme.

Animals↗
PubMed1988

Triosephosphate isomerase catalysis is diffusion controlled. Appendix: Analysis of triose phosphate equilibria in aqueous solution by 31P NMR.

The rates of the forward and reverse reactions of triosephosphate isomerase catalyzed by the wild-type and by a sluggish mutant enzyme have been studied in the absence and the presence of several viscosogenic agents. For the mutant enzyme, the kcat for which is some 10(3) times less than that for the wild-type enzyme, the value of kcat/Km with glyceraldehyde phosphate as substrate is almost unaffected by the presence of sucrose or glycerol, even though the concentration of the aldehyde form of the substrate is smaller because of hemiacetal formation. [The nature and relative amounts of the various forms of triose phosphate present in solution (free carbonyl forms, hydrates, dimers, hemiacetal adducts) have been evaluated by 31P NMR and are presented in the Appendix.] The viscosogenic agents cause the substrate to bind more tightly to the enzyme, roughly compensating for the lower substrate concentration. With dihydroxyacetone phosphate as substrate, the values of kcat/Km for the mutant enzyme increase with the addition of viscosogenic agent, consistent with the tighter binding of substrate without (in this case) any concomitant loss due to hemiketal formation. These results for the mutant enzyme (known to be limited in rate by an enolization step in the catalytic mechanism) can be used to interpret the behavior of the wild-type enzyme. Plots of the relative values of kcat/Km for catalysis by the wild-type enzyme (normalized with the corresponding data for the mutant enzyme) against the relative viscosity have slopes close to unity, as predicted by the Stokes-Einstein equation for a cleanly diffusive process. In the presence of polymeric viscosogenic additives such as poly(ethylene glycol), polyacrylamide, or ficoll, no effect on kcat/Km is seen for the wild-type enzyme, consistent with the expectation that molecular diffusion rates are unaffected by the macroviscosity and are only slowed by the presence of smaller agents that raise the microviscosity. These results show that the reaction catalyzed by the wild-type triosephosphate isomerase is limited by the rate at which glyceraldehyde phosphate encounters, or departs from, the active site.

Animals↗
PubMed1990

Proton diffusion in the active site of triosephosphate isomerase.

The current model for hydrogen flow in the aldose-ketose isomerases is probably incorrect. Enzymes of this class are characterized by both hydrogen transfer and proton exchange in the interconversion of substrate and product. The transfer is believed to be due to the action of a unique basic residue in the active site. Exchange is presumed to occur by dissociation of the abstracted proton and reassociation from the medium prior to its transfer to the intermediate enediol on the way to product. Dissociation of a necessary proton from the intermediate state imposes limits on the overall catalytic rate depending on the pKa of the protonated base and the pH of the medium. A case in point is triose-P isomerase (TIM), where kcat is approximately 10(4) s-1. T-Labeled substrate is found to lose approximately 95% of its T to the medium when totally converted to product. Although the active site base is believed to be a glutamate of pKa = 3.9, the pH dependence of maximum velocity is known to be flat up to pH 10. The loss of hydrogen required to form product as indicated by isotope exchange must be restored completely at this high pH, requiring a base of very high pKa, or there must be some other explanation for the loss of isotope. The present study demonstrates the existence of a single proton on human and rabbit TIM and three protons on yeast TIM that rapidly exchange with the abstracted proton at the E.enediol state internal exchange. Exchange with the medium external exchange occurs from the enzyme after substrate or product has dissociated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Crystallographic analysis of the complex between triosephosphate isomerase and 2-phosphoglycolate at 2.5-A resolution: implications for catalysis.

The binding of the transition-state analogue 2-phosphoglycolate to triosephosphate isomerase from yeast has been investigated crystallographically. An atomic model of the enzyme-inhibitor complex has been refined against data to 2.5-A resolution to a final R factor of 0.18. The interactions between the inhibitor and enzyme have been analyzed. The inhibitor forms hydrogen bonds to the side chains of His 95 and Glu 165. The latter hydrogen bond confirms that Glu 165 is protonated upon PGA binding. The structure of the complexed enzyme has been compared to that of the unbound form of the enzyme, and conformational changes have been observed: the side chain of Glu 165 moves over 2 A and a 10-residue flexible loop moves over 7 A to close over the active site. Spectroscopic results of phosphoglycolic acid binding to triosephosphate isomerase that have been amassed over the years are also explained in structural terms. The implications for catalysis are noted.

Binding Sites↗
PubMed1990

Combined effects of two mutations of catalytic residues on the ketosteroid isomerase reaction.

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by a conservative tautomeric transfer of the 4 beta-proton to the 6 beta-position with Tyr-14 as a general acid and Asp-38 as a general base [Kuliopulos, A., Mildvan, A. S., Shortle, D., & Talalay, P. (1989) Biochemistry 28, 149-159]. Primary, secondary, and combined deuterium kinetic isotope effects establish concerted substrate enolization to be the rate-limiting step with the wild-type enzyme [Xue, L., Talalay, P., & Mildvan, A. S. (1990) Biochemistry 29, 7491-7500]. The product of the fractional kcat values resulting from the Y14F mutation (10(-4.7)) and the D38N mutation (10(-5.6)) is comparable (10(-10.3)) to that of the double mutant Y14F + D38N (less than or equal to 10(-10.4)) which is completely inactive. Hence, the combined effects are either additive or synergistic. Quantitatively, similar effects of the two mutations on kcat/KM are found in the double mutant. Despite its inactivity, the Y14F + D38N double mutant forms crystals indistinguishable in form from those of the wild-type enzyme, tightly binds steroid substrates and substrate analogues, and immobilizes a spin-labeled steroid in an orientation indistinguishable from that found in the wild-type enzyme, indicating that the double mutant is otherwise largely intact. It is concluded that the total enzymatic activity of ketosteroid isomerase probably results from the independent and concerted functioning of Tyr-14 and Asp-38 in the rate-limiting enolization step, in accord with the perpendicular or antarafacial orientation of these two residues with respect to the enzyme-bound substrate. Synergistic effects of mutating two residues on kcat and on kcat/KM of enzyme-catalyzed multistep reactions are shown, theoretically, to occur when both residues act independently in the same step, and simple additivity occurs when this step is rate-limiting. Other conditions for additivity of the effects of mutations of kcat and kcat/KM are theoretically explored.

Binding Sites↗
PubMed1981

Concentration of activated intermediates of the fructose-1,6-bisphosphate aldolase and triosephosphate isomerase reactions.

As discovered by Grazi & Trombetta [Grazi, E., & Trombetta, G. (1978) Biochem. J. 175, 361], fructose-1,6-bisphosphate aldolase of rabbit muscle causes the slow formation of inorganic phosphate (Pi) and methylglyoxal when incubated with dihydroxyacetone phosphate (DHAP). In addition, these authors found an acid-labile intermediate in equilibrium with the aldolase-dihydroxyacetone phosphate complexes representing approximately 60% of the enzyme-bound DHAP species. Experiments are reported here which argue that this acid-labile species is the enzyme-bound enamine phosphate or its equivalent that decomposes by beta elimination in acid. A similar mechanism involving an enediol phosphate is proposed to explain a phosphatase action of triosephosphate isomerase that produces methylglyoxal and Pi at the rate of approximately 0.1 s(-1) at pH 5.5. When DHAP with excess isomerase is quenched in strong acid, the formation of Pi indicates that approximately 5% of bound reactant is in the form of enediol phosphate. The remainder of the substrate is about equally distributed between bound forms of DHAP and D-glyceraldehyde 3-phosphate. This equilibrium differs by 300-fold from the appropriate equilibrium in solution. Yeast aldolase, contrary to expectation, does not catalyze formation of inorganic phosphate and methylglyoxal when incubated with DHAP and gives no evidence fro an enediol phosphate intermediate when quenched in acid.

Animals↗
PubMed1981

Synthesis of 1-chloro-2-oxohexanol 6-phosphate, a covalent active-site reagent for phosphoglucose isomerase.

A new covalent active site reagent, 1-chloro-2-oxo-6-hexanol 6-phosphate, has been synthesized from glutaric acid monomethyl ester and characterized by NMR spectroscopy. Inactivation of phosphoglucose isomerase, when incubated with various modifier concentrations, was found to be pseudo first order with respect to enzyme concentration (half-life of inactivation 6 h at pH 7.5 (30 degrees C) and 2.0 muM active site concentration) but showed saturation kinetics for the dependence on inactivator concentration. This saturation phenomenon demonstrates the occurrence of a reversible enzyme-inhibitor complex (Kdiss = 14.2 mM) preceding the irreversible inactivation via the chloromethyl oxo groups. Substrate or competitive inhibitors such as 6-phosphogluconate or 5-phosphoarabinonate protect against inactivation of the isomerase by the modifying reagent.

Animals↗
PubMed2002

Receptor type I and type II binding regions and the peptidyl-prolyl isomerase site of cyclophilin B are required for enhancement of T-lymphocyte adhesion to fibronectin.

Cyclophilin B (CyPB), a cyclosporin A (CsA) binding protein, interacts with two types of binding sites at the surface of T-lymphocytes. The type I sites correspond to functional receptors involved in endocytosis and the type II sites to sulfated glycosaminoglycans (GAGs). Mutational analysis of CyPB has revealed that W128, which is part of the CsA-binding pocket, is implicated in the binding to the functional type I receptors and that two amino acid clusters located in the N-terminus ensure the binding to GAGs. The peptidyl-prolyl isomerase activity of CyPB is not required for receptor binding. We have recently demonstrated that CyPB enhances adhesion of peripheral blood T-lymphocytes to fibronectin, a component of the extracellular matrix. We intended to identify additional amino acids involved in the binding of CyPB to its functional type I receptor and to determine regions responsible for the stimulation of peripheral blood T-lymphocyte adhesion. We determined that residues R76, G77, K132, D155, and D158 of the calcineurin (CN) interacting region were implicated in the recognition of type I receptor but not of GAGs. We also found that two different changes in the N-terminal extension that abated binding to GAGs prevented adhesion of peripheral blood T-lymphocytes to coated CyPB, whereas abbrogation of the PPIase activity had no effect. On the other hand, the adhesion of peripheral blood T-lymphocytes to coated fibronectin was not stimulated by CyPB mutants devoid of either type I receptor or GAGs binding activity or by mutants of the PPIase site. Altogether, the results demonstrate that different regions of CyPB are involved in peripheral blood T-lymphocyte activation and imply a novel important physiological function for peptidyl-prolyl isomerase activity.

Adjuvants, Immunologic↗
PubMed2003

A complex of chaperones and disulfide isomerases occludes the cytosolic face of the translocation protein Sec61p and affects translocation of the prion protein.

Secretion of newly synthesized proteins across the mammalian rough endoplasmic reticulum (translocation) is supported by the membrane proteins Sec61p and TRAM, but may also include accessory factors, depending on the particular translocation substrate. Studies designed to investigate the binding of anti-peptide antibodies to the carboxyl terminus of the alpha-subunit of Sec61 (Sec61palpha) lead us to the isolation of a complex of proteins that occlude the cytosolic face of Sec61palpha in microsomes that have been prepared by standard protocols used to study translocation in vitro [Walter, P., and Blobel, G. (1983) Methods Enzymol. 96, 84-93]. This complex was shown by nanospray tandem mass spectrometry to be composed of protein disulfide isomerase (PDI), calcium binding protein 1 (CABP1/P5), 72 kDa endoplasmic reticulum protein (ERp72), and BiP (heat shock protein A5/HSPA5), and has been named TR-PDI for "translocon-resident protein disulfide isomerase complex". This constitutes a novel location for these proteins, which are known to be major constituents of the lumen of the rough endoplasmic reticulum. We have not established the function of TR-PDI at this location, but did observe that the absence of this complex results in a relative loss of correct topology of prion protein insertion across RER membranes, indicating the possibility of a functional role in vivo.

Amino Acid Sequence↗
PubMed2004

Crystal structure of human glutathione S-transferase A3-3 and mechanistic implications for its high steroid isomerase activity.

The crystal structure of human class alpha glutathione (GSH) S-transferase A3-3 (hGSTA3-3) in complex with GSH was determined at 2.4 A. Despite considerable amino acid sequence identity with other human class alpha GSTs (e.g., hGSTA1-1), hGSTA3-3 is unique due to its exceptionally high steroid double bond isomerase activity for the transformation of Delta(5)-androstene-3,17-dione (Delta(5)-AD) to Delta(4)-androstene-3,17-dione. A comparative analysis of the active centers of hGSTA1-1 and hGSTA3-3 reveals that residues in positions 12 and 208 may contribute to their disparate isomerase activity toward Delta(5)-AD. Substitution of these two residues of hGSTA3-3 with the corresponding residues in hGSTA1-1 followed by kinetic characterization of the wild-type and the mutant enzymes supported this prediction. On the basis of our model of the hGSTA3-3.GSH.Delta(5)-AD ternary complex and available biochemical data, we propose that the thiolate group of deprotonated GSH (GS(-)) serves as a base to initiate the reaction by accepting a proton from the steroid and the nonionized hydroxyl group of catalytic residue Y9 (HO-Y9) functions as part of a proton-conducting wire to transfer a proton back to the steroid. Residue R15 may function to stabilize the deprotonated thiolate group of GSH (GS(-)), and a GSH-bound water molecule may donate a hydrogen bond to the 3-keto group of Delta(5)-AD and thus help the thiolate of GS(-) to initiate the proton transfer and the subsequent stabilization of the reaction intermediate.

Amino Acid Substitution↗
PubMed2004

Isomerase-independent chaperone function of cyclophilin ensures aggregation prevention of adenosine kinase both in vitro and under in vivo conditions.

Using inactive aggregates of adenosine kinase (AdK) from Leishmania donovani as the model substrate, we recently demonstrated that a cyclophilin (LdCyP) from the same source in an isomerase-independent fashion reactivated the enzyme in vitro by disaggregating its inactive oligomers [Chakraborty et al. (2002) J. Biol. Chem. 277, 47451-47460]. Besides disrupting preformed aggregates, LdCyP also prevents reaggregation of the newly formed active protein that is generated after productive refolding from its urea-denatured state. To investigate possible physiological implications of such phenomena, a unique expression system that simultaneously induces both AdK and LdCyP in naturally AdK-deficient Escherichia coli, was developed. Both in vitro and in vivo experiments revealed that oligomerization is an inherent property of this particular enzyme. In vivo protein cross-linking studies, activity determination analysis and Ado phosphorylation experiments carried out in cells coexpressing both the proteins unequivocally demonstrated that, similar to the phenomena observed in vitro, aggregates of the enzyme formed in vivo are able to interact with both LdCyP and its N-terminal truncated form (N(22-88)DEL LdCyP) in a crowded intracellular environment, resulting in aggregation prevention and reactivation of the enzyme. Our results indicate that the isomerase-independent chaperone function of LdCyP, detected in vitro, participates in vivo as well to keep aggregation-prone proteins in a monomeric state. Furthermore, analogous to yeast/bacterial two-hybrid systems, development of this simple coexpression system may help in the confirmation of interaction of two proteins under simulated in vivo conditions.

Adenosine Kinase↗
PubMed2004

Xylose isomerase in substrate and inhibitor michaelis states: atomic resolution studies of a metal-mediated hydride shift.

Xylose isomerase (E.C. 5.3.1.5) catalyzes the interconversion of aldose and ketose sugars and has an absolute requirement for two divalent cations at its active site to drive the hydride transfer rates of sugar isomerization. Evidence suggests some degree of metal movement at the second metal site, although how this movement may affect catalysis is unknown. The 0.95 A resolution structure of the xylitol-inhibited enzyme presented here suggests three alternative positions for the second metal ion, only one of which appears positioned in a catalytically competent manner. To complete the reaction, an active site hydroxyl species appears appropriately positioned for hydrogen transfer, as evidenced by precise bonding distances. Conversely, the 0.98 A resolution structure of the enzyme with glucose bound in the alpha-pyranose state only shows one of the metal ion conformations at the second metal ion binding site, suggesting that the linear form of the sugar is required to promote the second and third metal ion conformations. The two structures suggest a strong degree of conformational flexibility at the active site, which seems required for catalysis and may explain the poor rate of turnover for this enzyme. Further, the pyranose structure implies that His53 may act as the initial acid responsible for ring opening of the sugar to the aldose form, an observation that has been difficult to establish in previous studies. The glucose ring also appears to display significant segmented disorder in a manner suggestive of ring opening, perhaps lending insight into means of enzyme destabilization of the ground state to promote catalysis. On the basis of these results, we propose a modified version of the bridged bimetallic mechanism for hydride transfer in the case of Streptomyces olivochromogenes xylose isomerase.

Aldose-Ketose Isomerases↗