Tyrosyl Residues Research Articles

Phosphorylation of Tyrosyl Residues 350/354 of the β-Adrenergic Receptor Is Obligatory for Counterregulatory Effects of Insulin

Insulin stimulates a loss of function and increased phosphotyrosine content of the beta 2-adrenergic receptor in intact cells, raising the possibility that the beta 2-receptor itself is a substrate for the insulin receptor tyrosine kinase. Phosphorylation of synthetic peptides corresponding to cytoplasmic domains of the beta 2-adrenergic receptor by the insulin receptor in vitro and peptide mapping of the beta 2-adrenergic receptor phosphorylated in vivo in cells stimulated by insulin reveal tyrosyl residues 350/354 and 364 in the cytoplasmic, C-terminal region of the beta 2-adrenergic receptor as primary targets. Mutation of tyrosyl residues 350, 354 (double mutation) to phenylalanine abolishes the ability of insulin to counterregulate beta-agonist stimulation of cyclic AMP accumulation. Phenylalanine substitution of tyrosyl reside 364, in contrast, abolishes beta-adrenergic stimulation itself.

Spin-Density Distribution, Conformation, and Hydrogen Bonding of the Redox-Active Tyrosine YZ in Photosystem II from Multiple-Electron Magnetic-Resonance Spectroscopies: Implications for Photosynthetic Oxygen Evolution

The oxidized form of the redox-active Y{sub Z} tyrosyl residue involved in photosynthetic oxygen evolution has been generated and trapped in Mn-depleted Photosystem II core complexes from a D2-Y160F mutant strain of Synechocystis 6803. This system eliminates interference from P{sub 700}{sup +} and Y{sub D}D+{center_dot} and allowed characterization of Y{sub Z}{sup {center_dot}} by using a combination of specific {sup 2}H-labeling and electron magnetic-resonance techniques that included CW-EPR, frequency-modulated and transient detected ENDOR, and {sup 2}H-ESEEM. Using these complementary techniques, we have carried out a detailed evaluation of the hyperfine structure of Y{sub Z}{sup {center_dot}} and obtained the dipolar interactions to weakly coupled nuclei, the strongly anisotropic tensors of the ring-hydrogens, and the more isotropic interactions to the {Beta}-methylene site. We conclude that tyrosyl radicals are not tuned to specific function by large-scale modulations of their spin density through hydrogen-bonding effects. We suggest a hydrogen-atom transfer function for Y{sub Z} in water oxidation. Within this model, the (Mn){sub 4}/Y{sub Z} center forms the Oxygen-Evolving Complex of Photosystem II where the (Mn){sub 4} cluster binds substrate water and delocalizes oxidizing equivalents and Y{sub Z} acts by abstracting hydrogens from substrate water in either a concerted or sequential fashion. 71 refs., 8 figs.,more » 2 tabs.« less

The action of philanthotoxin-343 and photolabile analogues on locust (Schistocerca gregaria) muscle.

The effects of philanthotoxin-343 (PhTX-343; tyrosyl-butanoyl-spermine) and photolabile analogues of this synthetic toxin on locust (Schistocerca gregaria) skeletal muscle have been investigated using whole muscle preparations (twitch contractions), single muscle fibres (excitatory postsynaptic currents (EPSCs)) and muscle membrane patches containing single quisqualate-sensitive glutamate receptors (qGluR). Analogues containing an azido group attached to either the butanoyl side-chain of PhTX-343 or as a substitute for the hydroxyl moiety of the tyrosyl residue were about 6 fold more potent antagonists than PhTX-343; those with an azido group located at the distal end of the toxin molecule were generally 2-3 fold less potent than PhTX-343. When these compounds were tested in subdued light, they were reversible antagonists of the muscle twitch, EPSC and qGluR. When a muscle was irradiated with U.V. during application of photolabile toxin combined with either neural stimulation of the muscle or L-glutamate application, antagonism of the twitch, EPSC and qGluR was complete and irreversible.

Structural features of the hydroxy- and keto-disubstituted bile salts: human serum albumin binding

The binding of keto- and hydroxy bile salts to human serum albumin, the identity of the bile salts binding sites and the identification of the amino acids present in these sites were studied. The keto bile salts cholanate-3-one (C3), cholanate-3,6-dione (C3-6) and cholanate-3-hydroxy-6-one (KHC) were found to quench the native fluorescence emission of albumin. This suggested that the tryptophan residue of human albumin (residue 214) is accessible to the keto bile salts and not to the hydroxy parent compounds. The binding of the keto bile salts was characterized by a simple population of binding sites with K a ranging from 22 · 10 4 M −1 for the mono keto bile salt (C3) down to 4 · 10 4 M −1 for the hydroxy-keto bile salt (KHC). The substitution of an oxo group at carbon C 3 in C3-6 molecule for a hydroxy group (KHC) produce a significant decreasing of the interaction, suggesting that the hybridization state of the carbon at C 3 in the steroid ring of the bile salt molecule is also an essential requirement for bile salts binding. It was found that bile salts are bound to the benzodiazepine binding site on human albumin (site II), producing a perturbation on site I, fatty acids and bilirubin binding site. The presence of only one substituent at C 3 (oxo or OH) produce an important perturbation on the fatty acid binding sites, decreasing the polarity of the its microenvironment, while a little effect was observed for the dihydroxy and di-oxo-substituted BS, suggesting that the hydroxy substituents at C 6, C 7 and C 12 do not interact in a significant manner with the fatty acid binding sites on HSA. The participationn of specific amino acids in albumin-bile salt binding sites depends on the polar groups on the bile salt molecules as exemplified by the quantitatively different role of lysyl residues like those interacting with KHC, C3 and C3-6, and tyrosyl residue interacting with KHC. The following amino acids in human albumin were found to play a role in the bile salts-albumin interaction: lysyl 195 and 225, several arginyls, histidyl 146 and tyrosyl 411.

Autoxidation of 4-Methylcatechol: A Model for the Study of the Biosynthesis of Copper Amine Oxidases Quinonoid Cofactor

The autoxidation of 4-methylcatechol under quasi-physiological conditions, leading to 2-hyddroxy-5-1,4-benzoquinone, was investigated. The effects of pH and metal ions were examined. An electrophilic attack of dioxygen to the 4-methylcatechol monoanion to form a transient peroxo species is proposed. It was concluded that such a non-enzymic conversion is likely for this model compound and for its physiological counterpart, a specific tyrosyl residue incorporated in the protein chain at the active site of copper amine oxidases.

Biochemical and Spectroscopic Characterization of the B800-850 Light-Harvesting Complex from Rhodobacter sulfidophilus and Its B800-830 Spectral Form

We demonstrate that the B800-830 spectral form of the B800-850 peripheral light-harvesting complex of Rhodobacter sulphidophilus, which is formed at low ionic strengths in the presence of the zwitterionic detergent LDAO, results from a local modification of the bacteriochlorophyll binding site and not the dissociation of the complex. This perturbation does not result in significant changes to the interactions between the pigments as studied by circular dichroism or fluorescence spectroscopy; however, modifications in the pigment binding sites are inferred from changes in the preresonance Raman spectrum. Specifically, an alteration of the hydrogen bonding of the 2-acetyl group of at least one of the bacteriochlorophyll groups that make up the 850 nm absorbing pair is observed. This implies an alteration in the conformation of the C-terminal domain of the alpha-polypeptide, in which are located the two tyrosyl residues that are believed to act as H-bond donors to these groups, induced by the protein-bound detergent in the absence of bound cations. We suggest that the ability of this complex to form an 800-830 complex is linked to the presence of an aspartyl residue immediately upstream of the tyrosyl residues. This study therefore provides a further illustration of the importance of hydrogen bonds to the 2-acetyl group of the bacteriochlorophyll in the determination of its spectral properties; furthermore, we provide a description of a conformational change that is able to modulate chromophore binding in these complexes.

Cloning, DNA Sequencing, and Amino Acid Sequencing of Catechol 1,2-Dioxygenases (Pyrocatechase) from Pseudomonas putida mt-2 and Pseudomonas arvilla C-1

Catechol 1,2-dioxygenase catalyzes the oxygenative ring cleavage of catechol to form cis,cis-muconic acid and is encoded by a catA gene, We have cloned a catA gene from Psudomonas putida mt-2 using a PCR product of amino acid sequence-based primers as a probe. The amino acid sequence deduced from the 930 nucleotides was in complete agreement with the chemically determined sequence of the protein. Crude extracts of Escherichia coli cells carrying the catA gene downstream from the lac promoter showed the enzyme activity. By using the same probe, we also cloned and sequenced the catA β gene for catechol 1,2-dioxygenase isozyme ββ from Pseudomonas arvilla C-1, which has three isozymes, αα, αβ, and ββ (C, Nakai, H. Horiike, S, Kuramitsu, H. Kagamiyama, and M. Nozaki, 1990, J. Biol. Chem. 265, 660-665). There was very high homology between isozyme ββ of the C-1 strain and the enzyme of the mt-2 strain in both the amino acid (98%) and the DNA sequences (97%). A preference for the use of codons terminating in C and G was found in the coding region of both the enzymes, which contributed to the high G + C content (65-66%) of the genes. A comparison of the DNA sequences of various catA genes from other sources revealed their common ancestry, whereas a comparison of the amino acid sequences of the enzymes revealed clear reflection of substrate specificity. Tyrosyl and histidyl residues for proposed ligands of ferric ion are conserved in all catechol 1,2-dioxygenases.

A sensitive fluorometric assay for protein-bound DOPA and related products of radical-mediated protein oxidation.

Oxidative attack on proteins results in the hydroxylation of tyrosyl residues to protein-bound DOPA (3,4-dihydroxyphenylalanine). Existing methods for assaying protein-bound DOPA have poor sensitivity and numerous possible interferences, such that accurate determination (especially of very low DOPA concentrations) has required time-consuming acid hydrolysis and HPLC analysis with fluorometric detection. This work presents a sensitive and selective assay for peptide or protein-bound o-benzoquinones derived from DOPA based on fluorometric detection of ethylenediamine derivatives. Detection limits for protein-bound DOPA are in tbe range 0.53-4.70 ng/mL for the assay mixture, corresponding to sample DOPA concentrations of 0.59-5.30 ng/mL (representing a minimum of 6-54 pmole detected), depending on the particular protein/peptide under study. The assay response increases linearly with DOPA concentration, and also with the extent of radical exposure of the protein. The assay is a simple and fast way to assess DOPA formation and thus oxidative damage in a protein.

Lipid peroxidation by peroxidase-catalyzed bioactivation of tyrosine.

Tyrosyl free radicals generated by the peroxidase-catalyzed oxidation of peptide tyrosyl residues are known to yield the stable cross-linked product dityrosine. In the present report, horseradish peroxidase is used as a model of peroxidase to study oxidative modifications of non-protein cellular components. Tyrosyl free radicals promote, as many free radicals, the decay of β-phycoerythrin fluorescence emission, they oxidize NADH and ascorbic acid and initiate arachidonic acid peroxidation with formation of hydroperoxides and dienes. These results suggest that tyrosyl free radicals generated when tyrosine residues in protein and peptides are activated in vivo by peroxidase-H2O2 might undergo the peroxidation of membrane lipids.

Conformational changes in pantetheine hydrolase as a function of guanidinium chloride concentration

The denaturation of pantetheinase (pantetheine hydrolase, EC 3.5.1.-) was followed in guanidinium chloride using tyrosyl and tryptophanyl residues as probes in connection with change in enzymatic activity. Movements of tryptophanyl and tyrosyl residues during denaturation were studied by second-derivative and fluorescence spectroscopy and the number of these amino acids present in the protein was calculated from spectroscopic data. Pantetheinase shows a very high resistance to denaturation, being completely unfolded at guanidinium chloride concentration higher than 6.5 M. Monitoring enzymatic activity shows that inactivation of the enzyme occurred before noticeable conformational changes were detected and it is suggested that the conformation of the active site is flexible and easily perturbable compared to the protein as a whole. This inactivation is reversible, as shown by renaturation experiments. Second-derivative and fluorescence spectra showed also that tyrosyl and tryptophanyl residues are largely exposed in the native protein, confirming its hydrophobic behavior.

Overexpression of the Protein Tyrosine Phosphatase, Nonreceptor Type 6 (PTPN6), in Human Epithelial Ovarian Cancer

Our current understanding of human ovarian tumorigenesis is limited by the lack of a discrete precursor lesion as well as a limited knowledge of the steps in tumor progression. Since the alterations in the regulation of the tyrosyl residues on various cellular proteins appear to be an important pathway in neoplastic transformation, it is possible that changes in the expression of the proteins that control tyrosine phosphorylation (i.e., tyrosine kinases and phosphatases) may play a role in ovarian cancer development. Protein tyrosine phosphatase, nonreceptor type 6 (PTPN6), contains two src homology 2 domains and is expressed primarily in hematopoietic and epithelial cells. Using Northern blot and immunoblotting analysis, we showed that both the PTPN6 transcripts and proteins were overexpressed two- to four-fold in 7 of the 8 ovarian epithelial carcinoma cell lines studied. In addition, we showed that there was also a two- to threefold increase in expression of the PTPN6 transcript in 10 of 11 (91%) invasive ovarian epithelial cancer tissues examined. These observations suggest that the PTPN6 gene is potentially of etiologic relevance to a majority of ovarian cancers.

DNA processing reactions in bacterial conjugation.

Bacterial conjugation is an important source of genetic plasticity. The initiation complex for conjugative transfer of transmissible plasmids--the relaxosome--is a specific DNA-protein structure that has been isolated from cells and reconstituted from purified components in vitro. Complexes containing uncleaved DNA and DNA cleaved at the nicsite in the origin of transfer (oriT) coexist in equilibrium. Relaxase is usually loaded onto oriT by accessory DNA-binding proteins. Relaxase catalyzes cleavage of a specific phosphodiester bond at nic and becomes covalently linked through a tyrosyl residue to the 5' terminus of the cleaved strand. Cleaved DNA may be unwound for transfer by a plasmid-encoded helicase. Single-strand transfer is thought to occur by a replicative rolling circle mechanism. Termination of a round of transfer is achieved by the cleaving-joining activity of the relaxase linked to the 5' end of the transferring strand. Relationships between DNA processing reactions and conjugative interactions of cell envelopes are particularly obscure aspects of the conjugation cycle.

Altered expression of protein-tyrosine phosphatase 2C in 293 cells affects protein tyrosine phosphorylation and mitogen-activated protein kinase activation.

PTP2C, an SH2 domain-containing protein-tyrosine phosphatase, is recruited to the growth factor receptors upon stimulation of cells. To investigate its role in growth factor signaling, we have overexpressed by approximately 6-fold the native PTP2C and a catalytically inactive mutant of the enzyme in 293 human embryonic kidney cells. The native PTP2C was located entirely in the cytosol, while the inactive mutant was nearly equally distributed in cytsolic and membrane fractions. Expression of the latter caused hyperphosphorylation on tyrosine of a 43-kDa protein, which was coimmunoprecipitated and co-partitioned in the plasma membrane fraction with the inactive PTP2C mutant. This protein may represent a physiological substrate of PTP2C. Overexpression of the native PTP2C enhanced epidermal growth factor (EGF)-stimulated mitogen-activated protein (MAP) kinase activity by 30%, whereas expression of the inactive mutant reduced the stimulated activity by 50%. Similar effects were observed for the activation of MAP kinase as determined by activity assay, gel mobility shift, and tyrosine phosphorylation. The data suggest that the phosphatase activity of PTP2C is partly required for MAP kinase activation by EGF and that PTP2C may function by dephosphorylating the 43-kDa membrane protein.

Chemical Modification of Leukotriene A4 Hydrolase. Indications for Essential Tyrosyl and Arginyl Residues at the Active Site

We have employed chemical modification to identify amino acids essential for the catalytic activities of the bifunctional zinc metalloenzyme leukotriene A4 hydrolase (EC 3.3.2.6). The epoxide hydrolase and the peptidase activity were both rapidly inactivated by N-acetylimidazole and tetranitromethane. Furthermore, treatment with 2,3-butanedione and phenylglyoxal also resulted in loss of both activities. Leukotriene A4 hydrolase could be protected from inactivation by these tyrosyl and arginyl reagents by the competitive inhibitors bestatin and captopril, respectively. Two tyrosyl and three arginyl residues were found by differential labeling techniques to be protected by the inhibitors, which thus suggested that these amino acids are located close to or at the active center of the enzyme. Limited modification by thiol reagents and particularly methyl methanethiosulfonate led to a > 10-fold increase in the peptidase activity and a decreased epoxide hydrolase activity, whereas prolonged treatment inhibited both activities. Kinetic analysis of modified enzyme, using the substrate alanine p-nitroanilide, revealed that the stimulatory effect on the peptidase activity was due to increased enzyme displayed a reduced apparent affinity constant for chloride ions, which strongly stimulate the peptidase activity. Neither activation nor inactivation by methyl methanethiosulfonate was influenced by the presence of competitive inhibitors, which suggested that this compound did not react with amino acids at the active center but rather with residues of importance for the overall enzyme conformation.

Copper/Topa Quinone-containing Histamine Oxidase from Arthrobacter globiformis: MOLECULAR CLONING AND SEQUENCING, OVERPRODUCTION OF PRECURSOR ENZYME, AND GENERATION OF TOPA QUINONE COFACTOR

The gene coding for histamine oxidase has been cloned and sequenced from a Coryneform bacterium Arthrobacter globiformis. The deduced amino acid sequence consists of 684 residues with a calculated molecular mass of 75,109 daltons and shows a high overall identity (58%) with that of phenethylamine oxidase derived from the same bacterial strain. Although the sequence similarities are rather low when compared with copper amine oxidases from other organisms, the consensus Asn-Tyr-Asp/Glu sequence, in which the middle Tyr is the precursor to the quinone cofactor (the quinone of 2,4,5-trihydroxyphenylalanine, topa) covalently bound to this class of enzymes, is also conserved in the histamine oxidase sequence. To identify the quinone cofactor, an overexpression plasmid has been constructed for the recombinant histamine oxidase. The inactive enzyme purified from the transformed Escherichia coli cells grown in a copper-depleted medium gained maximal activity upon stoichiometric binding of cupric ions. Concomitantly with the enzyme activation by copper, a brownish pink compound was generated in the enzyme, which was identified as the quinone of topa by absorption and resonance Raman spectroscopies of the p-nitrophenylhydrazine-derivatized enzyme and found at the position corresponding to the precursor Tyr (Tyr-402). Therefore, the copper-dependent autoxidation of a specific tyrosyl residue operates on the formation of the topa quinone cofactor in this enzyme, as recently demonstrated with the precursor form of phenethylamine oxidase (Matsuzaki, R., Fukui, T., Sato, H., Ozaki, Y., and Tanizawa, K. (1994) FEBS Lett. 351, 360-364).

Identification of an Essential Tyrosyl Residue in the Binding Site of Schizophyllum commune Xylanase A

Ultraviolet difference spectroscopy studies with the Schizophyllum commune xylanase in the presence of inhibitors and substrates indicated the participation of one or more tyrosyl residues in the binding of substrates to xylanase. Chemical modification experiments with group-specific reagents in the absence and presence of substrates confirmed the essential role of a tyrosyl residue in substrate binding while discounting the participation of tryptophan. A fourth-derivative absorbance spectroscopic method was developed to facilitate the quantitation of modified tyrosyl and tryptophanyl residues. This analysis showed that two tyrosyl residues of the xylanase are modified by tetranitromethane in the absence of substrate with the concomitant loss of catalytic activity. Protection of the xylanase with xylooligosaccharides resulted in the nitration of only one residue, and such enzyme derivatives retained 94% catalytic activity. Differential modification of the xylanase with tetranitromethane generated an enzyme derivative with the characteristic absorbance at 428 nm of 3-nitrotyrosine. Amino acid analysis and N-terminal sequencing of peptides with strong absorbance at 428 nm isolated from the protease-digested modified enzyme by reverse-phase HPLC identified the essential residue as Tyr97. Alignment of the S. commune xylanase amino acid sequence with those of the 18 other known family G xylanases revealed that Tyr97 is a conserved aromatic residue, further suggesting its essential role in substrate binding.

The proximal ligand variant His93Tyr of horse heart myoglobin

The spectroscopic and structural properties of the His93Tyr variant of horse heart myoglobin have been studied to assess the effects of replacing the proximal His residue of this protein with a tyrosyl residue as occurs in catalases from various sources. The variant in the ferric form exhibits electronic spectra that are independent of pH between pH 7 and 10, and it exhibits changes in absorption maxima and intensity that are consistent with a five-coordinate heme iron center at the active site. The EPR spectrum of the variant is that of a high-spin, rhombic system similar to that reported for bovine liver catalase. The 1D 1H-NMR spectrum of the variant confirms the five-coordinate nature of the heme iron center and exhibits a broad resonance at 112.5 ppm that is attributable to the meta protons of the phenolate ligand. This result indicates that the new Tyr ligand flips at a significant rate in this protein. The thermal stability of the Fe(III) derivative is unchanged from that of the wild-type protein (pH 8) while the midpoint reduction potential [-208 mV vs SHE (pH 8.0, 25 degrees C)] is about 250 mV lower. The three-dimensional structure of the variant determined by X-ray diffraction analysis confirms the five-coordinate nature of the heme iron center and establishes that the introduction of a proximal Tyr ligand is accommodated by a shift of the F helix (residues 88-99) in which this residue resides away from the heme pocket. Additional effects of this change are small shifts in the positions of Leu29, a heme propionate, and a heme vinyl group that are accompanied by altered hydrogen bonding interactions with the heme prosthetic group.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and conformation of the monohydrate of N-t-boctyrosyl-proline (Boc-Tyr-Pro·H2O)

The structure and conformation of the monohydrate of N-t-boc-tyrosyl-proline (Boc-Tyr-Pro·H2O) (C19H26O6N2·H2O) has been investigated with X-ray crystallographic and spectroscopic methods. Boc-Tyr-Pro crystallized in an extendedtrans conformation in the space group P212121 with cell dimensionsa=8.566(1),b=9.996(1),c=24.734(1). The conformation of Boc-Tyr-Pro reflex α-helix type prolines. Three intermolecular hydrogen bonds are observed. Crystal water is involved in two hydrogen bonds (to the hydroxyl group of the C-terminal of the proline residue; to the carbonyl group of the t-Boc functionality) while the hydroxyl group of the tyrosyl residue (to the carbonyl group of the amide bond) is involved in one hydrogen bond. The puckering mode of the pyrrolidine ring of the proline residue is similar to what has been previously observed for other proline-containing peptides.Cis-trans isomerism is observed in the NMR spectra of Boc-Tyr-Pro with a predominance for the extended side chain for the tyrosyl residue.

Synthesis and iodination of human (phenylalanine 13, tyrosine 19) melanin-concentrating hormone for radioreceptor assay.

An analogue of human melanin-concentrating hormone (MCH) suitable for radioiodination was designed in which Tyr13 and Val19 of the natural peptide were replaced by phenylalanyl and tyrosyl residues: [Phe13, Tyr19]-MCH. The peptide was synthesized by the continuous-flow solid-phase methodology using Fmoc-strategy and polyhipe PA 500 and PEG-PS resins. The linear MCH peptides with either acetamidomethyl-protected or free cysteinyl residues were purified to homogeneity and cyclized by iodine oxidation, yielding the final product with the correct molecular weight of 2434.61. Radioiodination of the C-terminal tyrosine was carried out enzymatically using solid-phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed-phase mini-column and by high-pressure liquid chromatography. The resulting [125I]-[Phe13, Tyr19]-MCH tracer was the first radiolabelled MCH peptide suitable for radioreceptor assay: saturation binding analysis using mouse G4F-7 melanoma cells demonstrated the presence of 1090 MCH receptors per cell. The dissociation constant (KD) was 1.18 x 10(-10) M, indicating high-affinity MCH receptors on these cells. MCH receptors were also found in other cell lines such as mouse B16-F1 and G4F and human RE melanoma cells as well as in PC12 and COS-7 cells. Competition binding analyses with a number of other peptides such as alpha-MSH, neuropeptide Y, substance P and pituitary adenylate cyclase activating peptide, demonstrated that the binding to the MCH receptor is specific. Atrial natriuretic factor was found to be a weak competitor of MCH, indicating topological similarities between MCH and ANF when interacting with MCH receptors.

DNA Processing Reactions in Bacterial Conjugation

Bacterial conjugation is an important source of genetic plasticity. The initiation complex for conjugative transfer of transmissible plasmids—the relaxosome—is a specific DNA-protein structure that has been isolated from cells and reconstituted from purified components in vitro. Complexes containing uncleaved DNA and DNA cleaved at the nicsite in the origin of transfer (oriT) coexist in equilibrium. Relaxase is usually loaded onto oriT by accessory DNA-binding proteins. Relaxase catalyzes cleavage of a specific phosphodiester bond at nic and becomes covalently linked through a tyrosyl residue to the 5′ terminus of the cleaved strand. Cleaved DNA may be unwound for transfer by a plasmid-encoded helicase. Single-strand transfer is thought to occur by a replicative rolling circle mechanism. Termination of a round of transfer is achieved by the cleaving-joining activity of the relaxase linked to the 5′ end of the transferring strand. Relationships between DNA processing reactions and conjugative interactions of cell envelopes are particularly obscure aspects of the conjugation cycle.