Chemical and enzymatic modification of proteins in the 30 S ribosome of Escherichia coli

Methods are described for the iodination of ribosomal proteins by iodine monochloride and potassium iodide and bovine lactoperoxidase. Ribosomes that were maximally iodinated did not synthesize polyphenylalanine. About one-half of the tyrosine residues could be iodinated with iodine monochloride in the intact ribosome with no change in the sedimentation properties of the particle. When proteins were extracted and dissolved in 5 m-urea, all of the tyrosine residues could be iodinated with iodine monoehloride.     

Mechanistic studies on carboxypeptidase A from goat pancreas Part I: Role of tyrosine residue at the active site

Chemical modification of carboxypeptidase Ag₁ from goat pancreas with Nacetylimidazole or iodine led to loss of enzymic activity. This loss in activity could be prevented when chemical modification was carried out in the presence of Β-phenylpropionic acid or substrate NCbz-glycyl-L-phenylalanine, thus suggesting a tyrosine residue at the active site. Chemical modification of tyrosine was confirmed by spectral and kinetic studies. While tyrosine modification destroyed peptidase activity, esterase activity of the enzyme remained unchanged thus indicating non-involvement of tyrosine residue in ester hydrolysis     

Neurospora crassa invertase. A study of amino acids at the active center.

1. The effects on Neurospora crassa invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) of a variety of group specific reagnets and other potential inhibitors were determined during a search for an irreversible inhibitor of the enzyme. Aniline, pyridoxal, enzyme substrate and products did not inactivate invertase under reducing conditions. Bromoacetic acid, iodoacetic acid, iodoacetamide, p-chloromercuribenzoate, hydroxylamine and 2-hydroxy-5-nitrobenzyl bromide were also ineffective. Iodine was the only reagent which irreversibly inhibited invertase. 2. Invertase was rapidly inactivated by low concentrations of iodine, indicating specific inhibition. However, the enzyme could not be protected from this inactivation by substrate. It was not reactivated by mercaptoethanol or cysteine. 3. Experiments on the uptake of radioactive iodine demonstrated that invertase is not iodinated under the conditions of iodine inactivation. 4. The sedimentation (S20,w) value of invertase was not altered by iodine inactivation. One-dimensional electrophoresis and finger-printing of tryptic digests revealed no differences between iodine treated and untreated invertase. There was no loss of carbohydrate from this glycoprotein during iodine inactivation. 5. Standard amino acid analyses of iodine-inactivated invertase showed some loss of tyrosine and a trace amount of methionine sulfone. Attempts to demonstrate oxidation of methionine to the sulfone, through modification of the procedure for preparation of samples for analysis, were unsuccessful. However, oxidation of half-cystine was indicated and further loss of tyrosine noted. A hypothesis is advanced that half-cystine is oxidized by iodine to a normally unstable oxidation state which is maintained and protected by its protein invironment and that loss of tyrosine may be an artifact caused by the presence of this residue during acid hydrolysis.     

Cloning, bacterial expression, purification, and characterization of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase

This report describes the cloning and characterization of rat leukocyte common antigen-related protein (rLAR), a receptor-like protein tyrosine phosphatase (PTPase). The recombinant cytoplasmic PTPase domain was expressed at high levels in bacteria and purified to homogeneity. Kinetic properties of the PTPase were examined along with potential modulators of PTPase activity. Several sulfhydryl-directed reagents were effective inhibitors, and a surprising distinction between iodoacetate and iodoacetamide was observed. The latter compound was an extremely poor inhibitor when compared to iodoacetate, suggesting that iodoacetate may interact selectively with a positive charge at or near the active site of the enzyme. Site-directed mutants were made at 4 highly conserved cysteine residues found at positions 1434, 1522, 1723, and 1813 within the protein. The Cys-1522/Ser mutation resulted in a 99% loss of enzymatic activity of the pure protein. This observation is consistent with greater than 99% of the PTPase activity being found in the first domain of the PTPase and demonstrates the critical importance of this cysteine residue in catalysis. The recombinant C1522S mutant phosphatase could also be phosphorylated in vitro by protein kinase C and p43v-abl tyrosine kinase. When pure recombinant PTPase was mixed with 32P-labeled tyrosine substrate and then rapidly denatured, a 32P-labeled enzyme intermediate could be trapped and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The catalytically inactive C1522S mutant did not form the phosphoenzyme intermediate.     

INACTIVATION OF PEPSIN BY IODINE AND THE ISOLATION OF DIIODO-TYROSINE FROM IODINATED PEPSIN

In the presence of iodine at pH 5.0–6.0 a solution of pepsin absorbs iodine and the specific proteolytic activity of the solution decreases. The activity is less than 1 per cent of the original activity when the number of iodine atoms per mol of pepsin is 35–40. If the pH is 4.5 or less, iodine reacts very slowly and there is a correspondingly slower loss in activity. Glycyl tyrosine reacts with iodine in a manner similar to pepsin. Experiments were performed to determine the extent to which oxidation of pepsin by iodine occurs during iodination, and if such oxidation were responsible for the loss in enzymatic activity. Although the results were not absolutely decisive, there seems to be no appreciable oxidation taking place during iodination and no relationship between the slight oxidation and loss in peptic activity. From a dialyzed preparation of completely iodinated pepsin which was inactive and contained 13.4 per cent bound iodine, 82 per cent of the iodine was obtained in a solution which analyzed as a solution of diiodo-tyrosine. Because of the presence of a material which contained no iodine and prevented quantitative crystallization, only 53 per cent of the iodine containing substance could be crystallized. This 53 per cent was, however, identified as diiodo-tyrosine. The part of the titration curve which in pepsin and most proteins represents the phenolic group of tyrosine was, in the curve for iodinated pepsin, shifted toward the acid region as expected. From these results, it appears that the loss in proteolytic activity of pepsin, when treated with iodine under the specified conditions, is due to the reaction of the iodine with the tyrosine in pepsin.     

A newly discovered oxidant defence system and its involvement in the development of Aurelia aurita (Scyphozoa, Cnidaria): reactive oxygen species and elemental iodine control medusa formation

In Aurelia aurita, applied iodine induces medusa formation (strobilation). This process also occurs when the temperature is lowered. This was found to increase oxidative stress resulting in an increased production of iodine from iodide. One polyp produces several medusae (initially termed ephyrae) starting at the polyp's oral end. The spreading of strobilation down the body column is controlled by a feedback loop: ephyra anlagen decrease the tyrosine content in adjacent polyp tissue by producing melanin from tyrosine. Endogenous tyrosine is able to remove iodine by forming iodiferous tyrosine compounds. The reduced level of tyrosine causes the ephyra-polyp-border to move towards the basal end of the former polyp. We argue that an oxidant defence system may exist which makes use of iodide and tyrosine. Like other marine invertebrates, polyps of Aurelia contain iodide ions. Inevitably produced peroxides oxidise iodide into iodine. The danger to be harmed by iodine is strongly decreased by endogenous tyrosine which reacts with iodine to form iodiferous tyrosine compounds including thyroxin. Both substances together, iodide and tyrosine, form an efficient oxidant defence system which shields the tissue against damage by reactive oxygen species. In the course of evolution (from a species at the basis of the animal kingdom like Aurelia to a highly evolved species like man) the waste product thyroxin (indicating a high metabolic rate) has developed into a hormone which controls the metabolic rate.     

On the mechanism of iodination of tyrosine

Existing data contain proof that the iodinating species of tyrosine and its derivatives contained in mixtures of iodine and iodide is hypoiodous acid, HOI. It appears likely that the peroxidase-catalyzed iodination reaction with hydrogen peroxide, tyrosine or a tyrosine derivative and either iodide or iodine as substrates involves enzyme-activated HOI.     

Dynamics and DNA substrate recognition by the catalytic domain of lambda integrase

Bacteriophage lambda integrase (lambda-Int) is the prototypical member of a large family of enzymes that catalyze site-specific DNA recombination via the formation of a Holliday junction intermediate. DNA strand cleavage by lambda-Int is mediated by nucleophilic attack on the scissile phosphate by a conserved tyrosine residue, forming an intermediate with the enzyme covalently attached to the 3'-end of the cleaved strand via a phosphotyrosine linkage. The crystal structure of the catalytic domain of lambda-Int (C170) obtained in the absence of DNA revealed the tyrosine nucleophile at the protein's C terminus to be located on a beta-hairpin far from the other conserved catalytic residues and adjacent to a disordered loop. This observation suggested that a conformational change in the C terminus of the protein was required to generate the active site in cis, or alternatively, that the active site could be completed in trans by donation of the tyrosine nucleophile from a neighboring molecule in the recombining synapse. We used NMR spectroscopy together with limited proteolysis to examine the dynamics of the lambda-Int catalytic domain in the presence and absence of DNA half-site substrates with the goal of characterizing the expected conformational change. Although the C terminus is indeed flexible in the absence of DNA, we find that conformational changes in the tyrosine-containing beta-hairpin are not coupled to DNA binding. To gain structural insights into C170/DNA complexes, we took advantage of mechanistic conservation with Cre and Flp recombinases to model C170 in half-site and tetrameric Holliday junction complexes. Although the models do not reveal the nature of the conformational change required for cis cleavage, they are consistent with much of the available experimental data and provide new insights into the how trans complementation could be accommodated.     

Structural and mechanistic basis for the activation of a low-molecular weight protein tyrosine phosphatase by adenine

Although the activation of low-molecular weight protein tyrosine phosphatases by certain purines and purine derivatives was first described three decades ago, the mechanism of this rate enhancement was unknown. As an example, adenine activates the yeast low-molecular weight protein tyrosine phosphatase LTP1 more than 30-fold. To examine the structural and mechanistic basis of this phenomenon, we have determined the crystal structure of yeast LTP1 complexed with adenine. In the crystal structure, an adenine molecule is found bound in the active site cavity, sandwiched between the side chains of two large hydrophobic residues at the active site. Hydrogen bonding to the side chains of other active site residues, as well as some water-mediated hydrogen bonds, also helps to fix the position of the bound adenine molecule. An ordered water was found in proximity to the bound phosphate ion present in the active site, held by hydrogen bonding to N3 of adenine and Odelta1 of Asp-132. On the basis of the crystal structure, we propose that this water molecule is the nucleophile that participates in the dephosphorylation of the phosphoenzyme intermediate. Solvent isotope effect studies show that there is no rate-determining transfer of a solvent-derived proton in the transition state for the dephosphorylation of the phosphoenzyme intermediate. Such an absence of general base catalysis of water attack is consistent with the stability of the leaving group, namely, the thiolate anion of Cys-13. Consequently, adenine activates the enzyme by binding and orienting a water nucleophile in proximity to the phosphoryl group of the phosphoenzyme intermediate, thus increasing the rate of the dephosphorylation step, a step that is normally the rate-limiting step of this enzymatic reaction.     

Evidence for a high-spin Fe(IV) species in the catalytic cycle of a bacterial phenylalanine hydroxylase

Phenylalanine hydroxylase is a mononuclear non-heme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate dioxygen for the hydroxylation of phenylalanine to tyrosine. Rapid-quench methods have been used to analyze the mechanism of a bacterial phenylalanine hydroxylase from Chromobacterium violaceum. Mo?ssbauer spectra of samples prepared by freeze-quenching the reaction of the enzyme-(57)Fe(II)-phenylalanine-6-methyltetrahydropterin complex with O(2) reveal the accumulation of an intermediate at short reaction times (20-100 ms). The Mo?ssbauer parameters of the intermediate (δ = 0.28 mm/s, and |ΔE(Q)| = 1.26 mm/s) suggest that it is a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(II) hydroxylases, including a tetrahydropterin-dependent tyrosine hydroxylase. Analysis of the tyrosine content of acid-quenched samples from similar reactions establishes that the Fe(IV) intermediate is kinetically competent to be the hydroxylating intermediate. Similar chemical-quench analysis of a reaction allowed to proceed for several turnovers shows a burst of tyrosine formation, consistent with rate-limiting product release. All three data sets can be modeled with a mechanism in which the enzyme-substrate complex reacts with oxygen to form an Fe(IV)═O intermediate with a rate constant of 19 mM(-1) s(-1), the Fe(IV)═O intermediate hydroxylates phenylalanine with a rate constant of 42 s(-1), and rate-limiting product release occurs with a rate constant of 6 s(-1) at 5 °C.     

Active site of the replication protein of the rolling circle plasmid pC194.

Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids. The effect of mutations was examined by an in vivo activity test. Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays. We conclude that all three amino acids have a catalytic role. Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases. We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate. The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step.     

SOME EFFECTS OF IODINE AND OTHER REAGENTS ON THE STRUCTURE AND ACTIVITY OF TOBACCO MOSAIC VIRUS

1. Denatured tobacco mosaic virus has a number of SH groups corresponding to its total sulfur content of 0.2 per cent. The SH groups were estimated by titration with ferricyanide, tetrathionate, and p-chloromercuribenzoate in guanidine hydrochloride solution and by reduction of the uric acid reagent in urea solution. 2. The SH groups of tobacco mosaic virus or their precursors can be abolished by reaction of the native form of the virus with iodine. 3. Tobacco mosaic virus whose SH groups have been oxidized beyond the S-S stage by iodine but whose tyrosine groups have not been converted into di-iodotyrosine groups still retains its normal biological activity as shown by the number of lesions it causes on Nicotiana glutinosa plants and by the characteristic disease produced in Turkish tobacco plants. 4. The inoculation of Turkish tobacco plants with active virus whose SH groups have been abolished by iodine results in the production of virus with the normal number of SH groups. 5. If enough iodine is added to tobacco mosaic virus or if the iodine reaction is carried out at a sufficiently high temperature, then the tyrosine groups are converted into di-iodotyrosine groups and the virus is inactivated. 6. Tobacco mosaic virus can be almost completely inactivated by iodoacetamide under conditions under which iodoacetamide reacts with few if any of the protein''s SH groups. 7. Tobacco mosaic virus is not inactivated by dilute p-chloromercuribenzoate.     

Electrostatic Suppression Allows Tyrosine Site-specific Recombination in the Absence of a Conserved Catalytic Arginine

The active site of the tyrosine family site-specific recombinase Flp contains a conserved catalytic pentad that includes two arginine residues, Arg-191 and Arg-308. Both arginines are essential for the transesterification steps of strand cleavage and strand joining in DNA substrates containing a phosphate group at the scissile position. During strand cleavage, the active site tyrosine supplies the nucleophile to form a covalent 3′-phosphotyrosyl intermediate. The 5′-hydroxyl group produced by cleavage provides the nucleophile to re-form a 3′-5′ phosphodiester bond in a recombinant DNA strand. In previous work we showed that substitution of the scissile phosphate (P) by the charge neutral methylphosphonate (MeP) makes Arg-308 dispensable during the catalytic activation of the MeP diester bond. However, in the Flp(R308A) reaction, water out-competes the tyrosine nucleophile (Tyr-343) to cause direct hydrolysis of the MeP diester bond. We now report that for MeP activation Arg-191 is also not required. In contrast to Flp(R308A), Flp(R191A) primarily mediates normal cleavage by Tyr-343 but also exhibits a weaker direct hydrolytic activity. The cleaved MeP-tyrosyl intermediate formed by Flp(R191A) can be targeted for nucleophilic attack by a 5′-hydroxyl or water and channeled toward strand joining or hydrolysis, respectively. In collaboration with wild type Flp, Flp(R191A) promotes strand exchange between MeP- and P-DNA partners. Loss of a catalytically crucial positively charged side chain can thus be suppressed by a compensatory modification in the DNA substrate that neutralizes the negative charge on the scissile phosphate.     

Iodination of peptidyl chloromethyl ketones for protease affinity labels

The specificity of peptidyl chloromethyl ketones has been used to label proteases in complex biological systems by incorporating tyrosine into the structure for eventual radioiodination. Contrary to results with iodination of proteins, a mild reagent, that is, one which iodinates at neutrality, was unsuitable, giving complex mixtures with poor reproducibility, apparently because of side reactions at the chloromethyl ketone group. On the other hand, iodine monochloride in acetic acid provided clean products. In the cases examined where a tyrosine residue was not appropriate for the specificity of the target protease, this residue was located well displaced from the primary specificity site. The resultant diiodotyrosine-containing derivatives were generally highly active as protease inhibitors. The p-aminobenzoyl group was used as an alternative to tyrosine as an iodinatable component.     

Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination

Cre recombinase is a prototypical member of the tyrosine recombinase family of site-specific recombinases. Members of this family of enzymes catalyze recombination between specific DNA sequences by cleaving and exchanging one pair of strands between the two substrate sites to form a 4-way Holliday junction (HJ) intermediate and then resolve the HJ intermediate to recombinant products by a second round of strand exchanges. Recently, hexapeptide inhibitors have been described that are capable of blocking the second strand exchange step in the tyrosine recombinase recombination pathway, leading to an accumulation of the HJ intermediate. These peptides are active in the lambda-integrase, Cre recombinase, and Flp recombinase systems and are potentially important tools for both in vitro mechanistic studies and as in vivo probes of cellular function. Here we present biochemical and crystallographic data that support a model where the peptide inhibitor binds in the center of the recombinase-bound DNA junction and interacts with solvent-exposed bases near the junction branch point. Peptide binding induces large conformational changes in the DNA strands of the HJ intermediate, which affect the active site geometries in the recombinase subunits.     

Reaction of hen egg-white lysozyme with tetranitromethane: a new side reaction, oxidative bond cleavage at glycine 104, and sequential nitration of three tyrosine residues

We found that the reaction of hen egg-white lysozyme with an equimolar amount of tetranitromethane (TNM) at pH 8.0 and room temperature yielded derivatives in which the N-C bond of Gly104 is oxidatively cleaved, and a mono-nitrotyrosine lysozyme in which Tyr23 is nitrated. This bond cleavage occurred more predominantly with a decrease in the nitration of Tyr23, when the reaction was carried out under more dilute conditions. A possible mechanism in which a phenoxyl radical of Tyr 23 (an intermediate of nitration) is involved was proposed for this oxidative bond cleavage. When lysozyme was reacted with a 10 times molar excess of TNM, in addition to a mono-nitrotyrosine lysozyme in which only Try23 is nitrated, a di-nitrotyrosine lysozyme in which Tyr20 and Tyr23 are both nitrated and a tri-nitrotyrosine lysozyme in which Tyr20, Tyr23, and Tyr53 are all nitrated were obtained. However, no other possible mono- or di-nitrotyrosine lysozymes could be isolated. Thus, it is concluded that the three tyrosine residues in lysozyme are essentially nitrated sequentially with TNM in the order of Tyr23, Tyr20, and Tyr53. Since the derivatives obtained here were all active, none of the three tyrosine residues or the residues around Gly104 are considered to be very important for the lysozyme activity.     

A chaperone-dependent GSK3beta transitional intermediate mediates activation-loop autophosphorylation

Glycogen synthase kinase 3 (GSK3), a key component of the insulin and wnt signaling pathways, is unusual, as it is constitutively active and is inhibited in response to upstream signals. Kinase activity is thought to be increased by intramolecular phosphorylation of a tyrosine in the activation loop (Y216 in GSK3beta), whose timing and mechanism is undefined. We show that GSK3beta autophosphorylates Y216 as a chaperone-dependent transitional intermediate possessing intramolecular tyrosine kinase activity and displaying different sensitivity to small-molecule inhibitors compared to mature GSK3beta. After autophosphorylation, mature GSK3beta is then an intermolecular serine/threonine kinase no longer requiring a chaperone. This shows that autoactivating kinases have adopted different molecular mechanisms for autophosphorylation; and for kinases such as GSK3, inhibitors that affect only the transitional intermediate would be missed in conventional drug screens.     

Regulation of a rat lung protein tyrosine kinase activity by reversible phosphorylation/dephosphorylation

Incubation of a partially purified protein tyrosine kinase from rat lung with Mg2+ and ATP resulted in about 10-15-fold activation of the enzyme activity as judged by the phosphorylation of poly(Glu:Tyr,4:1), an exogenous substrate. The activation was time dependent and was associated with the phosphorylation of a single protein band of 50 kDa. Phosphoamino acid analysis of the phosphorylated protein indicated that tyrosine was the amino acid being phosphorylated. Upon gel filtration on a Sephacryl S-200 column, the phosphorylated protein co-eluted with protein tyrosine kinase and ATP-binding activities, suggesting that all three activities are part of the same protein. In addition, pretreatment of the partially purified protein tyrosine kinase with alkaline phosphatase inhibited its enzyme activity which could be restored by reincubation with Mg2+ and ATP. These data suggest that a temporal relationship exists between the phosphorylation and the activation states of rat lung protein tyrosine kinase, and that the phospho- and dephospho- forms represent the active and inactive (or less active) forms, respectively, of the enzyme.     

The chemotactic response to PDGF-BB: evidence of a role for Ras

The PDGF receptor-beta mediates both mitogenic and chemotactic responses to PDGF-BB. Although the role of Ras in tyrosine kinase- mediated mitogenesis has been characterized extensively, its role in PDGF-stimulated chemotaxis has not been defined. Using cells expressing a dominant-negative ras, we find that Ras inhibition suppresses migration toward PDGF-BB. Overexpression of either Ras-GTPase activating protein (Ras-GAP) or a Ras guanine releasing factor (GRF) also inhibited PDGF-stimulated chemotaxis. In addition, cells producing excess constitutively active Ras failed to migrate toward PDGF-BB, consistent with the observation that either excess ligand or excess signaling intermediate can suppress the chemotactic response. These results suggest that Ras can function in normal cells to support chemotaxis toward PDGF-BB and that either too little or too much Ras activity can abrogate the chemotactic response. In contrast to Ras overexpression, cells producing excess constitutively active Raf, a downstream effector of Ras, did migrate toward PDGF-BB. Cells expressing dominant-negative Ras were able to migrate toward soluble fibronectin demonstrating that these cells retained the ability to migrate. These results suggest that Ras is an intermediate in PDGF- stimulated chemotaxis but may not be required for fibronectin- stimulated cell motility.     

Pseudomonas aeruginosa 4-amino-4-deoxychorismate lyase: spatial conservation of an active site tyrosine and classification of two types of enzyme

4-Amino-4-deoxychorismate lyase (PabC) catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of Gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å) crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5′-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp ² to sp ³ conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of the mechanism.