Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG₂) showed the highest immobilization capacity (8.2 mg SBP?g^?1 PANIG₂). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the K_m was conserved while the specific V_max dropped from 14.6 to 11.4 µmol min^?1 µg^?1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.     

Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG₂) showed the highest immobilization capacity (8.2 mg SBP g^-1 PANIG₂). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the K_m was conserved while the specific V_max dropped from 14.6 to 11.4 µmol min^-1 µg^-1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.     

Expression and characterization of soybean seed coat peroxidase in Escherichia coli BL21(DE3)

Soybean seed coat peroxidase (SBP) is a valuable enzyme having a broad variety of applications in analytical chemistry, biochemistry, and food processing. In the present study, the sscp gene (Gene ID: 548068) was optimized based on the preferred codon usage of Escherichia coli, synthesized, and expressed in E. coli BL21(DE3). SDS-PAGE and western blot analysis of this expressed protein revealed that its molecular weight is approximately 39?kDa. The effects of induction temperature, concentration of isopropyl-β-D-thiogalactoside and hemin, induction time, expression time were optimized to enhance SBP production with a maximum activity of 11.23?U/mL (8.64?U/mg total protein). Furthermore, the kinetics of enzyme-catalyzed reactions of recombinant protein was determined. When 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was used as substrate, optimum reaction temperature and pH of the enzyme were 85°C and 5.0, respectively. The effects of metal ions on the enzymatic reaction were also further investigated. The SBP was successfully expressed in E. coli BL21(DE3) which would provide a more efficient production strategy for industrial applications of SBP.     

Simultaneous purification and immobilization of soybean hull peroxidase with a dye attached to chitosan mini-spheres

Soybean hull peroxidase (EC 1.11.1.7, SBP) was simultaneously purified and immobilized by dye affinity chromatography with Reactive Blue 4 attached to chitosan mini-spheres. Under optimized conditions, 96% of SBP was adsorbed to the matrix. Under the most stringent condition, only 49% was desorbed, whereas 2 M NaCl failed to desorb a significant amount of SBP. This behaviour allowed proposing the dye matrix as a support to immobilize SBP from a crude extract. The pH of maximum activity shifted from 7 to 3–5. SBP gained thermostability after immobilization: after 5?h at 85?°C, the remaining activity was 54%, whereas that of the free enzyme was 31%. The optimum temperature for the immobilized SBP was 75?°C, whereas that of the free enzyme was 55?°C. After two months at 4?°C, the activity loss of the immobilized SBP was only 3%. Immobilized SBP removed 80% of 2-bromophenol from wastewater in 180?min and, after five cycles of use, the activity loss was only 12.8%.     

Kinetics of haem binding to human albumin and haemopexin: nonspecific interactions of haem with proteins

The kinetics of haem binding to human serum albumin and haemopexin were studied by means of the stopped flow technique. The reaction could be divided into three kinetically clearly distinguished steps: (1) extremely fast reaction of haem with nonspecific binding sites on the surface of the apoprotein molecule; this type of haem binding site seems to exist in proteins in general; (2) by meaas of equilibrium with its monomer, haem is transferred to the specific binding site; this second order reaction takes about 1–2 s, the reaction rate constant amounts to ≈10⁶ l mol^?1 s^?1 both for albumin and haemopexin: (3) conformational changes of haemoprotein molecule, accompanied by changes of absorption spectra in the Soret region; this series of slow monomolecular reactions takes about 20 min. These results are discussed in connection with the mechanism of haem transport from blood to liver cells.     

Modulation of human cytidine deaminase by specific aminoacids involved in the intersubunit interactions

An investigation was made of the role exerted by some residues supposed to be involved in the intersubunit interaction and also in the catalytic site of homotetrameric human cytidine deaminase (T-CDA). Attention was focused on Y33, Y60, R68, and F137 residues that are a part of a conserved region in most T-CDAs. Hence, a series of site-directed mutagenesis experiments was set up obtaining seven mutants: Y60G, Y33G, Y33F Y33S, F137A, R68G, and R68Q. Each active purified mutant protein was characterized kinetically, with a series of substrates and inhibitors, and the effect of temperature on enzyme activity and stability was also investigated. Circular dichroism (CD) experiments at different temperatures and in presence of small amounts of sodium dodecyl sulphate (SDS) were performed in all the soluble mutant CDAs. The results obtained by site-directed mutagenesis studies were compared to the crystallographic data of B. subtilis CDA and E. coli CDA and to molecular modeling studies previously performed on human CDA. The mutation of Y60 to glycine produced an enzyme with a more compact quaternary structure with respect to the wild-type; this mutation did not have a dramatic effect on cytidine deamination, but it slightly affected the binding with the substrate. None of the mutant CDAs in Y33 showed enzymatic activity; they existed only as monomers, indicating that this residue, located at the intersubunit interface, may be responsible for the correct folding of human CDA. The insertion of an alanine instead of phenylalanine at position 137 led to a soluble but completely inactive enzyme unable to form a tetramer, suggesting that F137 residue may be important for the assembling of the tetramer and also for the arrangement of the CDA active site. Finally, R68G and R68Q mutations revealed that the presence of the amino group seems to be important for the catalytic process but not for substrate binding, as already shown in B. subtilis CDA. The quaternary structure of R68Q was not affected by the mutation, as shown by the SDS-induced dissociation experiments and CD studies, whereas R68G dissociated very easily in presence of small amounts of SDS. These experiments indicated that in the human CDA, the side chain of arginine 68 involved in the catalytic process in one subunit active site might come from another subunit. The data obtained from these studies confirmed the presence of a complicated set of intersubunit interactions in the active site of human CDA, as shown in other T-CDAs.     

Structure and stability of icosahedral particles of a covalent coat protein dimer of bacteriophage MS2

Particles formed by the bacteriophage MS2 coat protein mutants with insertions in their surface loops induce a strong immune response against the inserted epitopes. The covalent dimers created by fusion of two copies of the coat protein gene are more tolerant to various insertions into the surface loops than the single subunits. We determined a 4.7‐Å resolution crystal structure of an icosahedral particle assembled from covalent dimers and compared its stability with wild‐type virions. The structure resembled the wild‐type virion except for the intersubunit linker regions. The covalent dimer orientation was random with respect to both icosahedral twofold and quasi‐twofold symmetry axes. A fraction of the particles was unstable in phosphate buffer because of assembly defects. Our results provide a structural background for design of modified covalent coat protein dimer subunits for use in immunization.     

褐色种皮大豆与其黄色种皮衍生亲本的表型及基因型比较

大豆种皮色在从野生大豆到栽培大豆的选择过程中逐渐由黑色变成黄色,是重要的形态标记,因此,大豆种皮色相关基因的研究无论是对进化理论研究还是育种实践都具有非常重要的意义。利用褐色种皮J1265-2大豆及其衍生亲本黄色种皮大豆J1265-1为材料,通过SSR引物扩增片段,检验遗传背景的异同,同时对控制种皮的候选基因GmF3’H进行扩增和测序分析。结果表明,褐色种皮和黄色种皮材料不仅用161对SSR分子标记检测没有发现差异,其褐色种皮候选基因GmF3’H的编码区及起始密码子上游1465 bp序列也是一致的。因此,证明褐色种皮J1265-2大豆与其衍生亲本黄色种皮大豆J1265-1为近等基因系,其控制褐色种皮的基因型与已报道的基因型不同。     

Purification, characterization and stability of barley grain peroxidase BP 1, a new type of plant peroxidase

The major peroxidase of barley grain (BP 1) has enzymatic and spectroscopic properties that are very differeant from those of other known plant peroxidases (EC 1.11.1.7) and can therefore contribute to the understanding of the many physiological functions ascribed to these enzymes. To study the structure-function relationships of this unique model peroxidase, large-scale and Jaboratory-scale purifications have been developed. The two batches of pure BP 1 obtained were identical in their enzymatic and spectral properties, and confirmed that BP 1 is different from the prototypical horseradish peroxidase isoenzyme C (HRP C). However, when measuring the specific activity of BP 1 at pH 4.0 in the presence of 1 m M C_aCl₂, the enzyme was as competent as HRP C at neutral pH towards a variety of substrates (m M mg⁻¹ min⁻¹): coniferyl alcohol (930±48), caffeic acid (795±53), ABTS (2,2'-azino-di-[3-ethyl-benzothiazoline-(6)-sulfonic acid]) (840±47), ferulic acid (415±20), p -coumaric acid (325±12), and guaiacol (58±3). The absorption spectrum of BP 1 is blue-shifted compared to that of HRP C with a Soret maximum of 399–402 nm, depending on pH. The prosthetic group was shown to be iron-protoporphyrin IX, which is characteristic of plant peroxidases. BP 1 is stable from pH 3 to 11, indicating that its unusual spectral characteristics do not result from enzyme instability. The thermostability is also normal with a melting temperature of 75°C at pH 6.6, and 67°C at pH 4.0 and 8.3. It is clear that the unusual properties of BP 1 are genuine, and reflect a novel regulation of plant peroxidase function.     

The role of quaternary interactions on the stability and activity of ascorbate peroxidase.

Point mutations at the dimer interface of the homodimeric enzyme ascorbate peroxidase (APx) were constructed to assess the role of quaternary interactions in the stability and activity of APx. Analysis of the APx crystal structure shows that Glu112 forms a salt bridge with Lys20 and Arg24 of the opposing subunit near the axis of dyad symmetry between the subunits. Two point mutants, E112A and E112K, were made to determine the effects of a neutral (alanine) and repulsive (lysine) mutation on dimerization, stability, and activity. Gel filtration analysis indicated that the ratio of the monomer to dimer increased as the dimer interface interactions went from attractive to repulsive. Differential scanning calorimetry (DSC) data exhibited a decrease in both the transition temperature (Tm) and enthalpy of unfolding (deltaHc) with Tm = 58.3 +/- 0.5 degrees C, 56.0 +/- 0.8 degrees C, and 53.0 +/- 0.9 degrees C and deltaHc = 245 +/- 29 kcal/mol, 199 +/- 38 kcal/mol, and 170 +/- 25 kcal/mol for wild-type APx, E112A, and E112K, respectively. Similar changes were observed based on thermal melting curves obtained by absorption spectroscopy. No change in enzyme activity was found for the E112A mutant, and only a 25% drop in activity was observed for the E112K mutant which demonstrates that the non-Michaelis Menten kinetics of APx is not due to the APx oligomeric structure. The cryogenic crystal structures of the wild-type and mutant proteins show that mutation induced changes are limited to the dimer interface including an alteration in solvent structure.     

Arabidopsis ATP A2 peroxidase. Expression and high-resolution structure of a plant peroxidase with implications for lignification

Lignins are phenolic biopolymers synthesized by terrestrial, vascular plants for mechanical support and in response to pathogen attack. Peroxidases have been proposed to catalyse the dehydrogenative polymerization of monolignols into lignins, although no specific isoenzyme has been shown to be involved in lignin biosynthesis. Recently we isolated an extracellular anionic peroxidase, ATP A2, from rapidly lignifying Arabidopsis cell suspension culture and cloned its cDNA. Here we show that the Atp A2 promoter directs GUS reporter gene expression in lignified tissues of transgenic plants. Moreover, an Arabidopsis mutant with increased lignin levels compared to wild type shows increased levels of ATP A2 mRNA and of a mRNA encoding an enzyme upstream in the lignin biosynthetic pathway. The substrate specificity of ATP A2 was analysed by X-ray crystallography and docking of lignin precursors. The structure of ATP A2 was solved to 1.45 Å resolution at 100 K. Docking of p-coumaryl, coniferyl and sinapyl alcohol in the substrate binding site of ATP A2 were analysed on the basis of the crystal structure of a horseradish peroxidase C-CN-ferulic acid complex. The analysis indicates that the precursors p-coumaryl and coniferyl alcohols are preferred by ATP A2, while the oxidation of sinapyl alcohol will be sterically hindered in ATP A2 as well as in all other plant peroxidases due to an overlap with the conserved Pro-139. We suggest ATP A2 is involved in a complex regulation of the covalent cross-linking in the plant cell wall.     

Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.     

Conformational strictness required for maximum activity and stability of bovine pancreatic ribonuclease A as revealed by crystallographic study of three Phe120 mutants at 1.4 Å resolution

The replacement of Phe120 with other hydrophobic residues causes a decrease in the activity and thermal stability in ribonuclease A (RNase A). To explain this, the crystal structures of wild-type RNase A and three mutants--F120A, F120G, and F120W--were analyzed up to a 1.4 A resolution. Although the overall backbone structures of all mutant samples were nearly the same as that of wild-type RNase A, except for the C-terminal region of F120G with a high B-factor, two local conformational changes were observed at His119 in the mutants. First, His119 of the wild-type and F120W RNase A adopted an A position, whereas those of F120A and F120G adopted a B position, but the static crystallographic position did not reflect either the efficiency of transphosphorylation or the hydrolysis reaction. Second, His119 imidazole rings of all mutant enzymes were deviated from that of wild-type RNase A, and those of F120W and F120G appeared to be "inside out" compared with that of wild-type RNase A. Only approximately 1 A change in the distance between N(epsilon2) of His12 and N(delta1) of His119 causes a drastic decrease in k(cat), indicating that the active site requires the strict positioning of the catalytic residues. A good correlation between the change in total accessible surface area of the pockets on the surface of the mutant enzymes and enthalpy change in their thermal denaturation also indicates that the effects caused by the replacements are not localized but extend to remote regions of the protein molecule.     

Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface

The stability of tetrameric malate dehydrogenase from the green phototrophic bacterium Chloroflexus aurantiacus (CaMDH) is at least in part determined by electrostatic interactions at the dimer-dimer interface. Since previous studies had indicated that the thermal stability of CaMDH becomes lower with increasing pH, attempts were made to increase the stability by removal of (excess) negative charge at the dimer-dimer interface. Mutation of Glu165 to Gln or Lys yielded a dramatic increase in thermal stability at pH 7.5 (+23.6 -- + 23.9 degrees C increase in apparent t(m)) and a more moderate increase at pH 4.4 (+4.6 -- + 5.4 degrees C). The drastically increased stability at neutral pH was achieved without forfeiture of catalytic performance at low temperatures. The crystal structures of the two mutants showed only minor structural changes close to the mutated residues, and indicated that the observed stability effects are solely due to subtle changes in the complex network of electrostatic interactions in the dimer-dimer interface. Both mutations reduced the concentration dependency of thermal stability, suggesting that the oligomeric structure had been reinforced. Interestingly, the two mutations had similar effects on stability, despite the charge difference between the introduced side-chains. Together with the loss of concentration dependency, this may indicate that both E165Q and E165K stabilize CaMDH to such an extent that disruption of the inter-dimer electrostatic network around residue 165 no longer limits kinetic thermal stability.     

Thioredoxin-like domain of human kappa class glutathione transferase reveals sequence homology and structure similarity to the theta class enzyme

Glutathione transferases (GSTs) are a superfamily of enzymes that play a vital functional role in the cellular detoxification process. They catalyze the conjugation of the thiol group of glutathione (GSH) to the electrophilic groups of a wide range of hydrophobic substrates, leading to an easier removal of the latter from the cells. The kappa class is the least studied one among various classes within the superfamily. We report here the expression, purification, and crystal structure of human kappa class GST (hGSTK), which has been determined by the multiple-isomorphous replacement method and refined to 1.93 A resolution. The overall structure of hGSTK is similar to the recently reported structure of kappa class GST from rat mitochondrion. Each subunit of the dimeric hGSTK contains a thioredoxin (TRX)-like domain and a helical domain. A molecule of glutathione sulfinate, an oxidized product of GSH, is found to bind at the G site of each monomer. One oxygen atom of the sulfino group of GSF forms a hydrogen bond with the hydroxyl group of the catalytic residue Ser16. The TRX-like domain of hGSTK shares 19% sequence identity and structure similarity with human theta class GST, suggesting that the kappa class of GST is more closely related to the theta class enzyme within the GST superfamily. The structure of the TRX-like domain of hGSTK is also similar to that of glutathione peroxidase (GPx), implying an evolutionary relationship between GST and GPx.     

Crystallographic and functional studies of a modified form of eosinophil-derived neurotoxin (EDN) with novel biological activities

The crystal structure of a post-translationally modified form of eosinophil-derived neurotoxin (EDN) with four extra residues on its N terminus ((-4)EDN) has been solved and refined at atomic resolution (1 A). Two of the extra residues can be placed unambiguously, while the density corresponding to two others is poor. The modified N terminus appears to influence the position of the catalytically important His129, possibly explaining the diminished catalytic activity of this variant. However, (-4)EDN has been shown to be cytotoxic to a Kaposi's sarcoma tumor cell line and other endothelial cell lines. Analysis of the structure and function suggests that the reason for cytotoxicity is most likely due to cellular recognition by the N-terminal extension, since the intrinsic activity of the enzyme is not sufficient for cytotoxicity and the N-terminal extension does not affect the conformation of EDN.     

Crystal structures of vegetative soybean lipoxygenase VLX-B and VLX-D, and comparisons with seed isoforms LOX-1 and LOX-3

The lipoxygenase family of lipid-peroxidizing, nonheme iron dioxygenases form products that are precursors for diverse physiological processes in both plants and animals. In soybean (Glycine max), five vegetative isoforms, VLX-A, VLX-B, VLX-C, VLX-D, VLX-E, and four seed isoforms LOX-1, LOX-2, LOX-3a, LOX-3b have been identified. In this study, we determined the crystal structures of the substrate-free forms of two major vegetative isoforms, with distinct enzymatic characteristics, VLX-B and VLX-D. Their structures are similar to the two seed isoforms, LOX-1 and LOX-3, having two domains with similar secondary structural elements: a beta-barrel N-terminal domain containing highly flexible loops and an alpha-helix-rich C-terminal catalytic domain. Detailed comparison of the structures of these two vegetative isoforms with the structures of LOX-1 and LOX-3 reveals important differences that help explain distinct aspects of the activity and positional specificity of these enzymes. In particular, the shape of the three branches of the internal subcavity, corresponding to substrate-binding and O(2) access, differs among the isoforms in a manner that reflects the differences in positional specificities.     

Trifluoroethanol-induced conformational change of tetrameric and monomeric soybean agglutinin: Role of structural organization and implication for protein folding and stability

2,2,2-Trifuoroethanol (TFE)-induced conformational structure change of a β-sheet legume lectin, soybean agglutinin (SBA) has been investigated employing its exclusive structural forms in quaternary (tetramer) and tertiary (monomer) states, by far- and near-UV CD, FTIR, fluorescence, low temperature phosphorescence and chemical modification. Far-UV CD results show that, for SBA tetramer, native atypical β-conformation transforms to a highly α-helical structure, with the helical content reaching 57% in 95% TFE. For SBA monomer, atypical β-sheet first converts to typical β-sheet at low TFE concentration (10%), which then leads to a nonnative α-helix at higher TFE concentration. From temperature-dependent studies (5–60 °C) of TFE perturbation, typical β-sheet structure appears to be less stable than atypical β-sheet and the induced helix entails reduced thermal stability. The heat induced transitions are reversible except for atypical to typical β-sheet conversion. FTIR results reveal a partial α-helix conversion at high protein concentration but with quantitative yield. However, aggregation is detected with FTIR at lower TFE concentration, which disappears in more TFE. Near-UV CD, fluorescence and phosphorescence studies imply the existence of an intermediate with native-like secondary and tertiary structure, which could be related to the dissociation of tetramer to monomer. This has been further supported by concentration dependent far-UV CD studies. Chemical modification with N-bromosuccinimide (NBS) shows that all six tryptophans per monomer are solvent-exposed in the induced α-helical conformation. These results may provide novel and important insights into the perturbed folding problem of SBA in particular, and β-sheet oligomeric proteins in general.     

Structure of a glutathione conjugate bound to the active site of aldose reductase

Aldose reductase (AR) is a monomeric NADPH-dependent oxidoreductase that catalyzes the reduction of aldehydes, ketones, and aldo-sugars. AR has been linked to the development of hyperglycemic injury and is a clinical target for the treatment of secondary diabetic complications. In addition to reducing glucose, AR is key regulator of cell signaling through it's reduction of aldehydes derived from lipoproteins and membrane phospholipids. AR catalyzes the reduction of glutathione conjugates of unsaturated aldehydes with higher catalytic efficiency than free aldehydes. The X-ray structure of human AR holoenzyme in complex with the glutathione analogue S-(1,2-dicarboxyethyl) glutathione (DCEG) was determined at a resolution of 1.94 A. The distal carboxylate group of DCEG's dicarboxyethyl moiety interacted with the conserved AR anion binding site residues Tyr48, His110, and Trp111. The bound DCEG's glutathione backbone adopted the low-energy Y-shape form. The C-terminal carboxylate of DCEG glutathione's glycine formed hydrogen bonds to Leu301 and Ser302, while the remaining interactions between DCEG and AR were hydrophobic, permitting significant flexibility of the AR and glutathione (GS) analogue interaction. The observed conformation and interactions of DCEG with AR were consistent with our previously published molecular dynamics model of glutathionyl-propanal binding to AR. The current structure identifies major interactions of glutathione conjugates with the AR active-site residues.