Mechanistic investigation of smart polymer-protein conjugates

Many affinity separation and diagnostic applications rely upon both capture and release steps. There is thus a need for methods to enhance the reversibility of biomolecular interactions. We have previously demonstrated that stimuli-responsive polymers can be used to gate biomolecular reactions when conjugated near the active site of proteins. Here we have used a new smart polymer, N,N-dimethyl acrylamide-co-4-phenylazophenylacrylate that has allowed a mechanistic investigation of the smart polymer switches. This polymer was conjugated via a vinyl sulfone terminus to cysteine residues of genetically engineered streptavidin mutant E116C, where the polymer is conjugated close to the biotin-binding site, and streptavidin mutant S139C, where the conjugation site is distant. The biotin binding switching activity was strongly dependent on conjugation position, as the E116C conjugate displayed a large thermal response while the S139C conjugate displayed only small effects. Kinetic measurements of biotin release demonstrated that the off-rate of biotin was unperturbed and that the thermally triggered release of biotin with the E116C conjugate was due to the blocking the reassociation of biotin. The addition of free polymer to purified E116C conjugates was also shown to increase the blocking and release properties of the switch. This effect was site dependent, suggesting that the conjugated polymers were directing a physical aggregation near the binding site that effectively enhanced the switching activity. These investigations provide mechanistic insight that can be utilized to design better molecular switches for a variety of stimuli-responsive polymer-protein conjugates.     

Characterization of morphine-glucose-6-phosphate dehydrogenase conjugates by mass spectrometry

A key characteristic of the analyte-reporter enzyme conjugate used in the enzyme-multiplied immunoassay technique (EMIT) is the inhibition of the conjugate enzyme upon anti-analyte antibody binding. To improve our understanding of the antibody-induced inhibition mechanism, we characterized morphine-glucose-6-phosphate dehydrogenase (G6PDH) conjugates as model EMIT analyte-reporter enzyme conjugates. Morphine-G6PDH conjugates were prepared by acylating predominantly the primary amines on G6PDH with morphine 3-glucuronide NHS ester molecules. In this study, morphine-G6PDH conjugates were characterized using a combination of methods, including tryptic digestion, immunoprecipitation, matrix-assisted laser desorption ionization mass spectrometry, and electrospray ionization tandem mass spectrometry. Twenty-six conjugation sites were identified. The identified sites all were found to be primary amines. The degree of conjugation was determined to be less than the number of conjugation sites, suggesting heterogeneity within the morphine-G6PDH conjugate population. Two catalytically important residues in the active site (K22 and K183) were among the identified conjugation sites, explaining at least partially the cause of loss of activity due to the coupling reaction.     

Photoswitching of ligand association with a photoresponsive polymer-protein conjugate

Light-regulated molecular switches that reversibly control biomolecular function could provide new opportunities for controlling activity in diagnostics, affinity separations, bioprocessing, therapeutics, and bioelectronics applications. Here we show that site-specific conjugation of light-responsive polymers near the biotin-binding pocket of streptavidin provides control of ligand binding affinity in response to UV and visible light irradiation. Two different light-responsive polymers were utilized that display opposite photoresponsive solubility changes under UV or visible (vis) light irradiation in aqueous solutions. At 40 degrees C, the N,N-dimethylacrylamide (DMA)-co-4-phenylazophenyl acrylate (AZAA) copolymer (DMAA) was soluble under UV irradiation and precipitated under visible light, while the DMA-co-N-4-phenylazophenyl acrylamide (AZAAm) copolymer (DMAAm) was soluble under visible irradiation and precipitated under UV light. Both polymers were synthesized with a vinyl sulfone terminus and conjugated to the Glu116Cys (E116C) streptavidin mutant via thiol coupling. The DMAA-streptavidin conjugate bound biotin efficiently when the polymer was in the soluble state under UV irradiation, but under visible irradiation, the polymer collapsed and blocked free biotin association. Furthermore, if biotin was allowed to bind when the polymer was in the soluble state under UV irradiation, then when the polymer was collapsed by visible light, the streptavidin released the bound biotin. The DMAAm-streptavidin conjugate showed the opposite response, with association of biotin allowed under visible light irradiation and blocked under UV irradiation. The photoresponses of the streptavidin conjugates thus correspond to the original photoresponsive phase transition properties of the polymer switches triggered by the cis-trans isomerization of the diazo chromophores.     

Improved procedure for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide.

The procedures for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide were improved in several respects as compared with the previous methods (Eur. J. Biochem. 62, 285--292, 1976; J. Immunol. 116, 1554--1560, 1976). Maleimide residues were efficiently introduced into antibodies under an atmosphere of nitrogen; the average number of maleimide residues introduced into IgG and Fab' antibodies were 0.78 (0.65--0.86) and 0.86 (0.80--0.95) per molecule, respectively. The conjugation with the enzyme was performed at 4 degrees C at pH 6.5 for 15 or more hours. The conjugates were almost completely separated from unreacted IgG and Fab' by gel filtration. When the recoveries of IgG, Fab', and beta-D-galactosidase in the conjugates were 23-29, 35-44, and 99%, respectively, the average numbers of IgG and Fab' molecules conjugated with the enzyme were 1.5-1.7 and 2.1-2.8 per molecule, respectively. There was no significant impairment of beta-D-galactosidase activity or the activity of anti-human IgG antibody to bind to human IgG upon conjugation. However, the conjugate preparation was heterogeneous, and one-third of each preparation consisted of aggregated conjugates less useful in sandwich enzymoimmunoassay than the remaining material. The conjugate with Fab' antibody gave lower control values in sandwich enzymoimmunoassay with silicone rubber as a solid phase than that with IgG antibody.     

Peroxidase and fluorescein isothiocyanate as antibody markers. A quantitative comparison of two peroxidase conjugates prepared with glutaraldehyde or periodate anda fluorescein conjugate.

Batches of rabbit anti-human immunoglobulin G antibodies were labeled either with horseradish peroxidase, using the two-step glutaraldehyde method or the periodate method, or with fluorescein isothiocyanate (FITC). The peroxidase conjugates were isolated by chromatography using two different gel types. The five types of conjugates thus obtained were standardized to the same amount of rabbit immunoglobulin G. The antibody activity, as estimated by means of single radial immunodiffusion and passive hemagglutination, and the enzyme activity, determined with orthodianisidine, were compared. The ultimate dilutions and absolute amounts of the five conjugates giving positive reactions were determined in direct and indirect immunohistochemical tests, using both cryostat sections of skin and the agarose bead model system. It appeared that during the peroxidase conjugation procedures there was a considerable loss of abtibody and enzyme activity, whereas in the FITC conjugation procedure the antibody activity remained intact. Neverthe less, peroxidase conjugates prepared with glutaraldehyde still gave positive staining reactions in equal or somewhat higher dilutions than the fluorescin conjugate did. The peroxidase conjugates prepared with periodate could not be diluted to the same extent. For the detection of antibodies by indirect immunohistochemical methods, the peroxidase conjugate, prepared with glutaraldehyde, was comparable to the FITC conjugate. The peroxidase conjugate, prepared with periodate, was less effective.     

Dendrimer versus linear conjugate: Influence of polymeric architecture on the delivery and anticancer effect of paclitaxel

The relative difference in polymeric architectures of dendrimer and linear bis(poly(ethylene glycol)) (PEG) polymer in conjugation with paclitaxel has been described. Paclitaxel, a poorly soluble anticancer drug, was covalently conjugated with PAMAM G4 hydroxyl-terminated dendrimer and bis(PEG) polymer for the potential enhancement of drug solubility and cytotoxicity. Both conjugates were characterized by 1NMR, HPLC, and MALDI/TOF. In addition, molecular conformations of dendrimer, bis(PEG), paclitaxel, and its polymeric conjugates were studied by molecular modeling. Hydrolysis of the ester bond in the conjugate was analyzed by HPLC using esterase hydrolyzing enzyme. In vitro cytotoxicity of dendrimer, bis(PEG), paclitaxel, and polymeric conjugates containing paclitaxel was evaluated using A2780 human ovarian carcinoma cells. Cytotoxicity increased by 10-fold with PAMAM dendrimer-succinic acid-paclitaxel conjugate when compared with free nonconjugated drug. Data obtained indicate that the nanosized dendritic polymer conjugates can be used with good success as anticancer drug carriers.     

Polyethylene glycol conjugates of methotrexate varying in their molecular weight from MW 750 to MW 40000: synthesis,characterization, and structure-activity relationships in vitro and in vivo

Poly(ethylene glycol)s (PEGs) are potential drug carriers for improving the therapeutic index of anticancer agents. In this work, the anticancer drug methotrexate (MTX) was activated with N,N'-dicyclohexylcarbodiimide (DCC) and coupled to amino group bearing PEGs of MW 750, 2000, 5000, 10 000, 20,000, and 40,000. First, the activation process of MTX with DCC in the presence and absence of N-hydroxysuccinimide was analyzed through HPLC. Preincubation of methotrexate with DCC alone at 0 degrees C proved to be favorable with respect to the amount of activated species and the formation of byproducts. MTX-PEG conjugates were synthesized according to this procedure, isolated through size-exclusion chromatography, and characterized through analytical HPLC, MALDI-TOF spectrometry, and gel permeation chromatography. In a cell-free assay, all of the drug polymer conjugates inhibited the target enzyme of MTX, dihydrofolate reductase (DHFR), to a similar extent, but were not as active as free MTX. Additionally, incubation of the MTX-PEG40000 conjugate for 6 days at 37 degrees C in phosphate buffered saline (pH 7.4), in cell-conditioned medium, or in human serum revealed no significant release of methotrexate. These results, taken together, indicate that release of MTX from polymer conjugates is not necessary for an effective interaction with the active site of dihydrofolate reductase. Evaluation of the in vitro cytotoxicity of the MTX-PEG conjugates in two adherent and three suspension human tumor cell lines revealed that the IC(50) values of the tested compounds increased with the size of the drug-polymer conjugates. The most effective compound tested in these assays was the free drug MTX itself (IC(50) value ranging from approximately 0.01 to 0.05 microM), while the IC(50) values of the polymer conjugates were higher (IC(50) value for MTX-PEG750, 2000 and 5000: approximately 0.6-3 microM; for MTX-PEG10000 and 20000: approximately 2-7 microM; and for MTX-PEG40000: > 6 microM). Subsequently, MTX-PEG5000, MTX-PEG20000, and MTX-PEG40000 were evaluated in a human mesothelioma MSTO-211H xenograft model, and their antitumor effects were compared with free methotrexate and the albumin conjugate MTX-HSA, a conjugate that is currently in phase II clinical trials. In contrast to the in vitro results, the high molecular weight MTX-PEG conjugates exhibited the highest in vivo antitumor activity: At a dose of 40 and 80 mg/kg MTX-PEG5000 was less active than MTX at its optimal dose of 100 mg/kg; MTX-PEG20000 at a dose of 40 mg/kg showed antitumor efficacy comparable to MTX, but MTX-PEG40000 at a dose of 20 mg/kg was superior to MTX and demonstrated antitumor activity of the same order as MTX-HSA (20 mg/kg).     

Analysis of active center in hyperthermophilic cellulase from Pyrococcus horikoshii

A hyperthermostable endoglucanase from Pyrococcus horikoshii with the capability of hydrolyzing crystalline cellulose was analyzed. A protein engineering study was carried out to obtain a reduced-size mutant. Five amino acid residues at both the N- and C-terminus were found to be removable without any loss of activity or thermal stability. Site-directed mutagenesis was also performed on R102, N200, E201, H297, Y299, E342, and W377, residues possibly involved in the active center or in the recognition and binding of a cellulose substrate. The activity of the resulting mutants was considerably decreased, confirming that the mutated residues were all important for activity. A reduced-size enzyme, as active as the wild-type endoglucanase, was successfully obtained, plus the residues critical for its activity and specificity were confirmed. Consequently, an engineered enzyme with a reduced size was obtained, and the amino acids essential for activity were confirmed by site-directed mutagenesis and comparison with a known three-dimensional structure.     

Reversible Blocking of Amino Groups of Octreotide for the Inhibition of Formation of Acylated Peptide Impurities in Poly(Lactide-co-Glycolide) Delivery Systems

The purpose of this study was to develop a novel method to inhibit the formation of acylated peptide impurities in poly(d,l-lactide-co-glycolide) (PLGA) formulations by reversely blocking the amino groups of octreotide with maleic anhydride (MA). Two mono-MA conjugates with different modification sites (N terminus and Lys residue) and di-MA conjugate of octreotide were prepared and isolated by reversed-phase high-performance liquid chromatography (RP-HPLC). The polymer interaction of peptides and the formation of acylated peptides were monitored by RP-HPLC. The stability of MA-octreotide conjugates in PLGA films was studied in 0.1 M phosphate buffer (pH 7.4) at 37°C. The conjugation of MA to octreotide substantially inhibited the interaction of peptide with PLGA polymer and the subsequent formation of acylated peptide impurities. The MA-octreotides were successfully converted to intact octreotide as pH drops by PLGA hydrolysis. In PLGA films, MA-octreotide also showed complete inhibition of peptide acylation. In conclusion, MA conjugation provides a viable approach for stabilizing peptides in PLGA delivery systems.     

Synthesis, cellular transport, and activity of polyamidoamine dendrimer-methylprednisolone conjugates

Dendrimers have emerged as promising multifunctional nanomaterials for drug delivery due to their well-defined size and tailorability. We compare two schemes to obtain methylprednisolone (MP)-polyamidoamine dendrimer (PAMAM-G4-OH) conjugate. Glutaric acid (GA) was used as a spacer to facilitate the conjugation. In scheme A, PAMAM-G4-OH was first coupled to GA and then further conjugated with MP to obtain PAMAM-G4-GA-MP conjugates. This scheme yields a lower conjugation ratio of MP, presumably because of lower reactivity and steric hindrance for the steroid at the crowded dendrimer periphery. In scheme B, this steric hindrance was overcome by first preparing the MP-GA conjugate, which was then coupled to the PAMAM-G4-OH dendrimer. The (1)H NMR spectrum of the conjugate from scheme B indicates a conjugation of 12 molecules of MP with the dendrimer, corresponding to a payload of 32 wt %. In addition, conjugates were further fluorescent-labeled with fluoroisothiocynate (FITC) to evaluate the dynamics of cellular entry. Flow cytometry and UV/visible spectroscopic analysis showed that the conjugate is rapidly taken up inside the cell. Fluorescence and confocal microscopy images on A549 human lung epithelial carcinoma cells treated with conjugates show that the conjugate is mostly localized in cytosol. MP-GA-dendrimer conjugate showed comparable pharmacological activity to free MP, as measured by inhibition of prostaglandin secretion. These conjugates can potentially be further conjugated with a targeting moiety to deliver the drugs to specific cells in vivo.     

Bacterially synthesized vertebrate calmodulin is a specific substrate for ubiquitination

Calmodulin purified from bacteria which express a cloned chicken calmodulin gene can be selectively conjugated with ubiquitin, using enzymes present in reticulocyte extracts. Analyses of peptide products generated from limited proteolytic digestion of the calmodulin conjugate containing a single ubiquitin indicate that lysine 115 on calmodulin is the site of linkage. This linkage site is identical to that previously reported for calmodulin purified from Dictyostelium discoideum. Substrate-dependent ATP hydrolysis by a partially purified ubiquitin conjugation enzyme system from reticulocyte extracts was used to determine the enzyme affinity to calmodulin. Km values of 7 and 9 microM were determined for dictyostelium and the bacterially expressed calmodulin, respectively. The bacterially expressed calmodulin, unlike the Dictyostelium protein, can also form conjugates containing a 2-5 molar ratio of ubiquitin but at a slower rate than that observed for conjugation at lysine 115. Results from these studies further support our hypothesis that the post-translational methylation of lysine 115 found in most forms of calmodulin serves the important function of protecting calmodulin from ubiquitination and from degradation by the cytoplasmic ubiquitin-dependent proteolytic pathway. The capability of the bacterially expressed calmodulin to form conjugates with a high molar ratio of ubiquitin suggests that the post-translational acetylation of the N terminus of calmodulin may serve a similar function.     

Reversibly soluble biocatalyst: optimization of trypsin coupling to Eudragit S-100 and biocatalyst activity in soluble and precipitated forms

Eudragit S-100, a copolymer of methacrylic acid and methyl methacrylate is soluble at pH above 5 and insoluble at pH below 4.5. pH-dependent solubility of the polymer is used for the development of reversibly soluble biocatalyst, which combines the advantages of both soluble and immobilized biocatalysts. Activity of trypsin, covalently coupled to Eudragit S-100, was improved by protecting the active site of the enzyme with benzamidine and removing the noncovalently bound proteins with Triton X-100 in 0.15 M Tris buffer (pH 7.6). Accurate choice of coupling conditions combined with proper washing protocol produced highly active enzyme-polymer conjugate with no noncovalently bound protein. Two conjugates with 100-fold difference in the content of trypsin coupled to Eudragit S-100 were studied when the preparations were in soluble and precipitated forms. The K(m)values of the soluble enzyme to the lower molecular weight substrate was less than that of the free enzyme, whereas that to the higher molecular weight substrate was closer to that of the free enzyme. Activities of the soluble and precipitated immobilized trypsin with higher molecular weight substrate were completely inhibited by soy bean trypsin inhibitor, whereas complete inhibition with soy bean trypsin inhibitor was never achieved with lower molecular weight substrate, indicating reduced access of high-molecular weight substrate/inhibitor to some of the catalytically active enzyme molecules in trypsin-Eudragit conjugate.     

Activity studies of immobilized subtilisin on functionalized pure cellulose-based membranes

The activity of immobilized subtilisin BPN' on pure cellulose-based membrane support was investigated using site-directed and random immobilization approaches. The catalytic activity of site-directed immobilized subtilisin on pure cellulose fiber-based materials was found to be 81% of that in homogeneous solution, while that of randomly immobilized subtilisin was 27%. Pure cellulose membrane supports provided large surface areas for high enzyme loading without diffusional limitations. The activity of immobilized subtilisin on pure cellulose support was more than twice that on a modified polyether sulfone (MPS) membrane, which was attributed to the higher hydrophilicity of cellulose. Immobilized subtilisin maintained its initial activity for 14 days at 4 degrees C and 7 days at 24 degrees C. The immobilized enzyme could resist higher temperature and operate over a wider range of pH without loss of activity. This study showed that pure cellulose fiber-based membranes are well suited for enzyme immobilization and biocatalysis.     

Reduced immunogenicity of beta-lactoglobulin by conjugating with chitosan

Bovine beta-lactoglobulin (beta-LG) was conjugated with chitosan (CHS) by means of a water-soluble carbodiimide to reduce the immunogenicity of beta-LG. Each beta-LG-CHS conjugate was purified by ion-exchange chromatography and hydrophobic chromatography. The conjugation between beta-LG and CHS was confirmed by SDS-PAGE, the isoelectric point of the conjugate being higher than that of beta-LG. Two types of the beta-LG-CHS conjugate were obtained with molar ratios of beta-LG to CHS of 1:1 (F1) and 1:2 (F2). Structural analyses by fluorescence measurement, ELISA with monoclonal antibodies and retinol-binding activity indicated that the conjugates had almost maintained the native structure of beta-LG. The antigenicity of the beta-LG-CHS conjugates was similar to that of beta-LG in C3H/He mice. Reduction of the immunogenicity of beta-LG was achieved by conjugation with CHS. In particular, F2 showed very low immunogenicity. B cell epitopes of beta-LG and the conjugates recognized in C3H/He mice were determined with 15-mer multi-pin peptide; the linear epitope profiles of the conjugates were found to be similar to those of beta-LG, while the antibody response to each epitope was dramatically reduced. Conjugation of beta-LG with chitosan was effective for reducing the immunogenicity of beta-LG.     

Synthesis and characterization of a catalytic antibody-HPMA copolymer-Conjugate as a tool for tumor selective prodrug activation

Selective chemotherapy remains a key issue for successful treatment in cancer therapy. The use of targeting approaches like the enhanced permeability and retention (EPR) effect of macromolecules, is consequently needed. Here, we report the preparation of a novel catalytic antibody-polymer conjugate for selective prodrug activation. HPMA copolymer was conjugated to catalytic antibody 38C2 through an amide bond formation between epsilon-amino group of lysine residue from the antibody molecule and a p-nitrophenyl ester of the polymer. The conjugate was purified over a size exclusion column using FPLC. In the isolated fraction, one or two molecules of polymer were conjugated to one molecule of antibody based on gel analysis. The resulting conjugate retained most of its catalytic activity (75-81%) in comparison to the free antibody. The activity was monitored with a fluorogenic substrate and a prodrug activation assay using HPLC. Furthermore, the conjugate was evaluated in vitro for its ability to activate an etoposide prodrug using two different cancer cell lines. Cells growth inhibition using the prodrug and the conjugate was almost identical to inhibition by the free antibody and the prodrug. For the first time, a catalytic antibody was conjugated to a passive targeting moiety while retaining its catalytic ability to activate a prodrug. The conjugate described in this work can be used for selective activation of prodrug in the PDEPT (polymer directed enzyme prodrug therapy) approach by replacing the enzyme component with catalytic antibody 38C2.     

Functional stabilization of trypsin by conjugation with beta-cyclodextrin-modified carboxymethylcellulose

Bovine pancreatic trypsin was chemically modified by a beta-cyclodextrin-carboxymethylcellulose polymer using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as coupling agent. The conjugate retained 110% and 95% of the initial esterolytic and proteolytic activity, respectively, and contained about 2 mol of polymer per mol of trypsin. The optimum temperature for trypsin was increased to 8 degrees C after conjugation. The thermostability of the enzyme was increased to about 16 degrees C after modification. The conjugate prepared was also more stable against thermal incubation at different temperatures ranging from 45 degrees C to 60 degrees C. In comparison with native trypsin, the polymer-enzyme complex was more resistant to autolytic degradation at pH 9.0, retaining about 65% of the initial activity after 3h incubation. In addition, modification protected trypsin against denaturation in the presence of sodium dodecylsulfate.     

Bordetella pertussis fimbriae are effective carrier proteins in Neisseria meningitidis serogroup C conjugate vaccines

Serogroup C meningococcal conjugate vaccines generally use diphtheria or tetanus toxoids as the protein carriers. The use of alternative carrier proteins may allow multivalent conjugate vaccines to be formulated into a single injection and circumvent potential problems of immune suppression in primed individuals. Bordetella pertussis fimbriae were assessed as carrier proteins for Neisseria meningitidis serogroup C polysaccharide. Fimbriae were conjugated to the polysaccharide using modifications of published methods and characterised by size exclusion chromatography; co-elution of protein and polysaccharide moieties confirmed conjugation. The conjugates elicited boostable IgG responses to fimbriae and serogroup C polysaccharide in mice, and IgG:IgM ratios indicated that the responses were thymus-dependent. High bactericidal antibody titres against a serogroup C strain of N. meningitidis were also observed. In a mouse infection model, the conjugate vaccine protected against lethal infection with N. meningitidis. Therefore, B. pertussis fimbriae are effective carrier proteins for meningococcal serogroup C polysaccharide and could produce a vaccine to protect against meningococcal disease and to augment protection against pertussis.     

Influence of agonists and antagonists on the segmental motion of residues near the agonist binding pocket of the acetylcholine-binding protein

Using the Lymnaea acetylcholine-binding protein as a surrogate of the extracellular domain of the nicotinic receptor, we combined site-directed labeling with fluorescence spectroscopy to assess possible linkages between ligand binding and conformational dynamics. Specifically, 2-[(5-fluoresceinyl)aminocarbonyl]ethyl methanethiosulfonate was conjugated to a free cysteine on loop C and to five substituted cysteines at strategic locations in the subunit sequence, and the backbone flexibility around each site of conjugation was measured with time-resolved fluorescence anisotropy. The sites examined were in loop C (Cys-188 using a C187S mutant), in the beta9 strand (T177C), in the beta10 strand (D194C), in the beta8-beta9 loop (N158C and Y164C), and in the beta7 strand (K139C). Conjugated fluorophores at these locations show distinctive anisotropy decay patterns indicating different degrees of segmental fluctuations near the agonist binding pocket. Ligand occupation and decay of anisotropy were assessed for one agonist (epibatidine) and two antagonists (alpha-bungarotoxin and d-tubocurarine). The Y164C and Cys-188 conjugates were also investigated with additional agonists (nicotine and carbamylcholine), partial agonists (lobeline and 4-hydroxy,2-methoxy-benzylidene anabaseine), and an antagonist (methyllycaconitine). With the exception of the T177C conjugate, both agonists and antagonists perturbed the backbone flexibility of each site; however, agonist-selective changes were only observed at Y164C in loop F where the agonists and partial agonists increased the range and/or rate of the fast anisotropy decay processes. The results reveal that agonists and antagonists produced distinctive changes in the flexibility of a portion of loop F.     

Further characterization of the putative human isopeptidase T catalytic site

The human isopeptidase T (isoT) is a zinc-binding deubiquitinating enzyme involved in the disassembly of free K48-linked polyubiquitin chains into ubiquitin monomers. The catalytic site of this enzyme is thought to be composed of Cys335, Asp435, His786 and His795. These four residues were site-directed mutagenized. None of the mutants were able to cleave a peptide-linked ubiquitin dimer. Similarly, C335S, D435N and H795N mutants had virtually no activity against a K48-linked isopeptide ubiquitin dimer, which is an isoT-specific substrate that mimics the K48-linked polyubiquitin chains. On the other hand, the H786N mutant retained a partial activity toward the K48-linked substrate, suggesting that the His786 residue might not be part of the catalytic site. None of the mutations significantly affected the capacity of isoT to bind ubiquitin and zinc. Thus, the catalytic site of UBPs could resemble that of other cysteine proteases, which contain one Cys, one Asp and one His.     

A single amino acid in the PSPG-box plays an important role in the catalytic function of CaUGT2 (Curcumin glucosyltransferase), a Group D Family 1 glucosyltransferase from Catharanthus roseus

Curcumin glucosyltransferase (CaUGT2) isolated from cell cultures of Catharanthus roseus exhibits unique substrate specificity. To identify amino acids involved in substrate recognition and catalytic activity of CaUGT2, a combination of domain swapping and site-directed mutagenesis was carried out. Exchange of the PSPG-box of CaUGT2 with that of NtGT1b (a phenolic glucosyltransferase from tobacco) led to complete loss of enzyme activity in the resulting recombinant protein. However, replacement of Arg378 of the NtGT1b PSPG-box with cysteine, the corresponding amino acid in CaUGT2, restored the catalytic activity of the chimeric enzyme. Further site-directed mutagenesis revealed that the size of the amino acid side-chain in that particular site is critical to the catalytic activity of CaUGT2.