Analysis of the role of cysteine residues in isopenicillin N synthetase activity by site-directed mutagenesis.

The predicted amino acid sequences of isopenicillin N synthetase from both Cephalosporium acremonium and Penicillium chrysogenum have two cysteine residues in analogous positions (Cys-106 and Cys-255 in the C. acremonium numbering). To examine the role of these cysteine residues in the activity of the C. acremonium enzyme, we used site-directed in vitro mutagenesis to change these cysteine residues to serine residues. Mutation of Cys-255 reduces specific activity approximately equal to 50%, whereas mutation of Cys-106 or mutation of both Cys-106 and Cys-255 reduces specific activity about 97%. This suggests that the cysteines are important but not essential for IPNS activity. Alkylation of IPNS also almost completely inactivated the enzyme, but residual activity could have been due to incomplete alkylation. Atomic substitution via genetic manipulation in this case is a more accurate means of assessing the role of sulfhydryl moieties in enzyme activity.     

Argininosuccinate synthetase of bovine liver: chemical and physical properties.

Homogeneous crystalline argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5] prepared from bovine liver according to Rochovansky et al. [Rochovansky, O., Kodowaki, H. & Ratner, S. (1977)J. Biol. Chem. 252, 5287-5294] has been characterized with respect to amino acid composition and other chemical and physical properties. The total residue molecular weights derived from the amino acid analysis are in agreement with values previously obtained by physical means for the catalytically active tetramer, 185,000, and the monomer, 46,500. The enzyme is focused sharply at pH 7.6 as a single protein. Additional properties reported include 2.74 X 10(5) M-1 cm-1 for the molar absorption coefficient, based on the absolute value for protein, and 0.747 ml/g for the chemically based partial specific volume.     

Functional arginyl residues as ATP binding sites of glutamine synthetase and carbamyl phosphate synthetase.

The reaction of phenylglyoxal with two enzymes in which ATP plays a complex role has been studied. Both ovine brain glutamine synthetase and Escherichia coli carbamyl phosphate synthetase [carbamoyl-phosphate synthase (glutamine); ATP:carbamate phosphotransferase (dephosphorylating, amido-transferring); EC 2.7.2.9]were inactivated by phenylglyoxal. The specificity of this reagent for arginyl residues of the two proteins was confirmed by amino acid analysis. ATP, but not the other substrates, protected these enzymes against inactivation by phenylglyoxal. Carbamyl phosphate synthetase was also protected by IMP and ornithine, positive allosteric effectors that alter the enzymatic activity be increasing the affinity for ATP. UMP, a negative allosteric effector that decreases the affinity for ATP, did not protect against inactivation. Differential labeling experiments with [14C]phenylglyoxal showed that the number of arginyl residues protected by ATP corresponded quite well to the known number of ATP catalytic sites for each protein. These data indicate that arginyl residues at the active sites of glutamine synthetase and carbamyl phosphate synthetase are involved in the binding of ATP. This phenylglyoxal inactivation study also provided information about the mechanistic role of ATP in the two synthetases. The data obtained on glutamine synthetase support the theory that ATP is attached to the enzyme as a portion of the catalytic site, and that its presence is essential for the binding of glutamate and glutamine. The data obtained on carbamyl phosphate synthetase are consistent with the previous proposal that carbonyl phosphate is an intermediate in the ATP-dependent activation of bicarbonate by this enzyme. It is also of interest that, with both glutamine synthetase and carbamyl phosphate synthetase, only a small portion of the total arginyl population of these enzymes reacted with phenylglyoxal. A summary of previous studies on the modification of enzyme arginyl residues is presented.     

Ribonucleolytic activity of angiogenin: essential histidine, lysine, and arginine residues.

The homology of angiogenin and pancreatic RNase A provides a compelling reason to systematically compare the characteristics of the two proteins using the chemical modification approaches that proved essential to understanding the action of RNase. Reagents specific for histidine, lysine, and arginine markedly decrease the ribonucleolytic activity of angiogenin, much as has been observed for RNase A. Activity is abolished by reduction of the disulfide bonds and is restored by reoxidation. Methionine, tyrosine, and carboxyl group reagents have no significant effect. From the point of view of reactivity, the histidine and lysine residues in angiogenin are severalfold less susceptible to modification than those in RNase A. Arginine reagents, on the other hand, inactivate angiogenin considerably faster than RNase A. Considering specificity, bromoacetate inactivates angiogenin at pH 5.5 by modifying 1.5 histidines, but lysine and arginine reagents are less specific. Thus, 3.8 and 6.3 residues, respectively, are modified by 1-fluoro-2,4-dinitrobenzene and by formaldehyde plus cyanoborohydride, under conditions where activity decreases by approximately 80% in both cases. With phenylglyoxal, 6.7 arginines are lost when there is 92% inactivation. Poly(G) prevents inactivation by lysine and arginine reagents, and phosphate protects against the effects of lysine modification. Thus, the functional consequences of these modifications likely reflect the loss of critical residues rather than general conformational effects.     

Dimerization of thiol-specific antioxidant and the essential role of cysteine 47.

Thiol-specific antioxidant (TSA) from yeast contains cysteine residues at amino acid positions 47 and 170 but is not associated with obvious redox cofactors. These two cysteines are highly conserved in a family of proteins that exhibit sequence identity of 23-98% with TSA. The roles of Cys-47 and Cys-170 in yeast TSA were investigated by replacing them individually with serine and expressing the mutant TSA proteins (RC47S and RC170S, respectively), as well as wild-type TSA (RWT), in Escherichia coli. Wild-type TSA purified from yeast (YWT) and RWT were both shown to exist predominantly as dimers, whereas RC47S and RC170S existed mainly as monomers under a denaturing condition. This observation suggests that the dimerization of YWT and RWT requires disulfide linkage of Cys-47 and Cys-170. The presence of the Cys-47-Cys-170 linkage in YWT was directly shown by isolation of dimeric tryptic peptides, one monomer of which contained Cys-47 and the other contained Cys-170. A small percentage of YWT, RWT, RC47S, and RC170S molecules formed dimers linked by Cys-47-Cys-47 or Cys-170-Cys-170 disulfide bonds. The antioxidant activity of the various TSA proteins was evaluated from their ability to protect glutamine synthetase against the dithiothreitol/Fe3+/O2 oxidation system. YWT, RWT, and RC170S were equally protective, whereas RC47S was completely ineffective. Thus, Cys-47, but not Cys-170, constitutes the site of oxidation by putative substrate.     

Extracellular ATP in T-lymphocyte activation: possible role in effector functions.

We hypothesized that cytolytic T lymphocytes (CTL) may utilize extracellular ATP (ATPo) during the effector phase of the CTL-target cell interactions and that CTL could be the source of ATPo. It is demonstrated here that incubation of CTL with activating ligands [Con A or monoclonal antibody (mAb) to the T-cell antigen receptor (TCR)] results in the extracellular Ca2(+)-independent accumulation of the ATPo. The addition of the ATP-degrading enzymes into the mixture of CTL and target cells results in a strong inhibition of the CTL-mediated, TCR-triggered lethal-hit delivery to the target cell. In a parallel control experiment, the employed enzymes did not affect target cell-induced, TCR-triggered exocytosis of granules from CTL. Thus, the removal of ATPo with enzymes does not interfere with the activation of CTL by the target cell but does block lytic events. Cloned helper T lymphocytes also accumulate ATPo after incubation with anti-TCR mAb or Con A, suggesting the possibility that ATPo, which acts in concert with ectoprotein kinases and/or purinergic receptors, may be of general use as a messenger in cellular interactions of T lymphocytes.     

Cysteamine revisited: repair of arginine to cysteine mutations

Cysteamine is a small aminothiol endogenously derived from coenzyme A degradation. For some decades, synthetic cysteamine has been employed for the treatment of cystinosis, and new uses of the drug continue to emerge. In this review, we discuss the role of cysteamine in cellular and extracellular homeostasis and focus on the potential use of aminothiols to reconstitute the function of proteins harboring arginine (Arg) to cysteine (Cys) mutations, via repair of the Cys residue into a moiety that introduces an amino group, as seen in basic amino acid residues Lys and Arg. Cysteamine has been utilized in vitro and ex vivo in four different genetic disorders, and thus provides “proof of principle” that aminothiols can modify Cys residues. Other aminothiols such as mercaptoethylguanidine (MEG) with closer structural resemblance to the guanidinium moiety of Arg are under examination for their predicted enhanced capacity to reconstitute loss of function. Although the use of aminothiols holds clinical potential, more studies are required to refine specificity and treatment design. The efficacy of aminothiols to target proteins may vary substantially depending on their specific extracellular and intracellular locations. Redox potential, pH, and specific aminothiol abundance in each physiological compartment are expected to influence the reactivity and turnover of cysteamine and analogous drugs. Upcoming research will require the use of suitable cell and animal models featuring Arg to Cys mutations. Since, in general, Arg to Cys changes comprise about 8% of missense mutations, repair of this specific mutation may provide promising avenues for many genetic diseases.     

Testing a mechanism of control in human cholesterol metabolism: relation of arginine and glycine to insulin and glucagon

Eight men were given 2 casein meals, one with and one without a supplement of arginine and glycine, to measure the effect on plasma amino acids, insulin and glucagon. Supplementation resulted in increased levels of plasma glucagon, glycine and arginine, a tendency to decreased insulin and significantly lower insulin/glucagon ratio, tryptophan and tyrosine. The data suggest that insulin and glucagon, which control cholesterol metabolism, respond to dietary and postprandial plasma amino acid levels of arginine and glycine.     

Mutations affecting conserved cysteine residues within the extracellular domain of Neu promote receptor dimerization and activation.

Overexpression of the Neu/ErbB-2 receptor tyrosine kinase has been implicated in the genesis of human breast cancer. Indeed, expression of either activated or wild-type neu in the mammary epithelium of transgenic mice results in the induction of mammary tumors. Previously, we have shown that many of the mammary tumors arising in transgenic mice expressing wild-type neu occur through somatic activating mutations within the neu transgene itself. Here we demonstrate that these mutations promote dimerization of the Neu receptor through the formation of disulfide bonds, resulting in its constitutive activation. To explore the role of conserved cysteine residues within the region deleted in these altered Neu proteins, we examined the transforming potential of a series of Neu receptors in which the individual cysteine residues were mutated. These analyses indicated that mutation of certain cysteine residues resulted in the oncogenic activation of Neu. The increased transforming activity displayed by the altered receptors correlated with constitutive dimerization that occurred in a disulfide bond-dependent manner. We further demonstrate that addition of 2-mercaptoethanol to the culture medium interfered with the specific transforming activity of the mutant Neu receptors. These observations suggest that oncogenic activation of Neu results from constitutive disulfide bond-dependent dimerization.     

Multiple modes of activation of latent human fibroblast collagenase: evidence for the role of a Cys73 active-site zinc complex in latency and a "cysteine switch" mechanism for activation.

Latent human fibroblast collagenase (HFC) can be activated by a variety of seemingly disparate means. In addition to the well-characterized activation by trypsin and organomercurial compounds, the enzyme can be activated to various extents by surfactants such as sodium dodecyl sulfate, by chaotropic ions such as SCN-, by disulfide compounds such as oxidized glutathione, by sulfhydryl alkylating agents such as N-ethylmaleimide, and by oxidants such as NaOCl. The underlying basis for these activations is the modification, exposure, or proteolytic release of the Cys73 residue from its habitat in the latent enzyme where it is thought to be complexed to the active-site zinc atom. This residue is not accessible for reaction with small molar excesses of dithionitrobenzoate in native, latent HFC. However, on addition of EDTA, this residue becomes fully exposed and is quantitatively labeled. All modes of activation of latent HFC are believed to involve the dissociation of Cys73 from the active-site zinc atom and its replacement by water, with the concomitant exposure of the active site. This is thought to be the primary event that precedes the well-known autolytic cleavages that are observed following the appearance of collagenase activity. The dissociation of Cys73 from the zinc atom in the latent enzyme "switches" the role of the zinc from a noncatalytic to a catalytic one. This "cysteine switch" mechanism of regulation may be applicable to the entire collagenase gene family.     

Mutagenesis of essential functional residues in acetylcholinesterase.

The cholinesterases are serine hydrolases that show no global similarities in sequence with either the trypsin or the subtilisin family of serine proteases. The cholinesterase superfamily includes several esterases with distinct functions and other proteins devoid of the catalytic serine and known esterase activity. To identify the residues involved in catalysis and conferring specificity on the enzyme, we have expressed wild-type Torpedo acetylcholinesterase (EC 3.1.1.7) and several site-directed mutants in a heterologous system. Mutation of serine-200 to cysteine results in diminished activity, while its mutation to valine abolishes detectable activity. Two conserved histidines can be identified at positions 425 and 440 in the cholinesterase family; glutamine replacement at position 440 eliminates activity whereas the mutation at 425 reduces activity only slightly. The assignment of the catalytic histidine to position 440 defines a rank ordering of catalytic residues in cholinesterases distinct from trypsin and subtilisin and suggests a convergence of a catalytic triad to form a third, distinct family of serine hydrolases. Mutation of glutamate-199 to glutamine yields an enzyme with a higher Km and without the substrate-inhibition behavior characteristic of acetylcholinesterase. Hence, modification of the acidic amino acid adjacent to the serine influences substrate association and the capacity of a second substrate molecule to affect catalysis.     

An essential regulatory role for macrophage migration inhibitory factor in T-cell activation.

The protein known as macrophage migration inhibitory factor (MIF) was one of the first cytokines to be discovered and was described 30 years ago to be a T-cell-derived factor that inhibited the random migration of macrophages in vitro. A much broader role for MIF has emerged recently as a result of studies that have demonstrated it to be released from the anterior pituitary gland in vivo. MIF also is the first protein that has been identified to be secreted from monocytes/macrophages upon glucocorticoid stimulation. Once released, MIF acts to "override" or counter-regulate the suppressive effects of glucocorticoids on macrophage cytokine production. We report herein that MIF plays an important regulatory role in the activation of T cells induced by mitogenic or antigenic stimuli. Activated T cells produce MIF and neutralizing anti-MIF antibodies inhibit T-cell proliferation and interleukin 2 production in vitro, and suppress antigen-driven T-cell activation and antibody production in vivo. T cells also release MIF in response to glucocorticoid stimulation and MIF acts to override glucocorticoid inhibition of T-cell proliferation and interleukin 2 and interferon gamma production. These studies indicate that MIF acts in concert with glucocorticoids to control T-cell activation and assign a previously unsuspected but critical role for MIF in antigen-specific immune responses.     

Cysteine residues 110 and 187 are essential for the formation of correct structure in bovine rhodopsin.

To investigate the role of different cysteine residues in bovine rhodopsin, a series of mutants were prepared in which the cysteine residues were systematically replaced by serines. The mutant genes were expressed in monkey kidney cells (COS-1) and the mutant opsins were evaluated for their levels of expression, glycosylation patterns, and ability to form the chromophore characteristic of rhodopsin and to activate transducin. Substitution of the three cytoplasmic cysteines (Cys-316, Cys-322, and Cys-323) and the four membrane-embedded cysteines (Cys-140, Cys-167, Cys-222, and Cys-264) produced proteins with wild-type phenotype. Also, single substitutions of Cys-185 gave rise to a wild-type phenotype. In contrast, substitution of the three intradiscal cysteines (Cys-110, Cys-185, and Cys-187) or single substitution of Cys-110 or Cys-187 gave proteins that were expressed at reduced levels, glycosylated abnormally, and unable to bind 11-cis-retinal. Thus, of the 10 cysteines in bovine rhodopsin, only intradiscal Cys-110 and Cys-187 are essential for the correct tertiary structure of the protein.     

The structure of nonvertebrate actin: implications for the ATP hydrolytic mechanism

The structures of Saccharomyces cerevisiae, Dictyostelium, and Caenorhabditis elegans actin bound to gelsolin segment-1 have been solved and refined at resolutions between 1.9 and 1.75 A. These structures reveal several features relevant to the ATP hydrolytic mechanism, including identification of the nucleophilic water and the roles of Gln-137 and His-161 in positioning and activating the catalytic water, respectively. The involvement of these residues in the catalytic mechanism is consistent with yeast genetics studies. This work highlights both structural and mechanistic similarities with the small and trimeric G proteins and restricts the types of mechanisms responsible for the considerable enhancement of ATP hydrolysis associated with actin polymerization. The conservation of functionalities involved in nucleotide binding and catalysis also provide insights into the mechanistic features of members of the family of actin-related proteins.     

Siderophore electrochemistry: relation to intracellular iron release mechanism.

Previous studies have shown that there is a major difference between the iron release mechanism of enterobactin, a catechol-based siderophore, and that of the hydroxamate-based siderophores such as ferrichrome. For ferric enterobactin there is an esterase that hydrolyzes the ligand during iron release. In contrast, iron is released by the hydroxamate-based siderophores and the ligands are reused in subsequent iron transport. It has been suggested that release of iron by hydroxamates occurs by reduction to the ferrous complex, a process that does not occur for ferric enterobactin. Cyclic voltammograms of ferrichrome A and ferrioxamine B exhibit reversible one-electron waves with pH-independent formal potentials (Ef-vs. the normal hydrogen electrode) -446 and -454 mV, respectively, within the range of physiological reductants. Ferric enterobactin also shows a reversible one-electron wave (at pH greater than 10) with Ef = -986 mV vs. the normal hydrogen electrode. From the pH dependence of this potential we estimate a reduction potential of -750 mV at pH 7. In sharp contrast to the value for the ferric hydroxamates, this value is well below the range of physiological reducing agents. The results demonstrate that the observed hydrolysis of enterobactin is a necessary prerequisite to in vivo release of iron from the siderophore via ferric ion reduction.     

The folding mechanism of larger model proteins: role of native structure.

The folding mechanism of a 125-bead heteropolymer model for proteins is investigated with Monte Carlo simulations on a cubic lattice. Sequences that do and do not fold in a reasonable time are compared. The overall folding behavior is found to be more complex than that of models for smaller proteins. Folding begins with a rapid collapse followed by a slow search through the semi-compact globule for a sequence-dependent stable core with about 30 out of 176 native contacts which serves as the transition state for folding to a near-native structure. Efficient search for the core is dependent on structural features of the native state. Sequences that fold have large amounts of stable, cooperative structure that is accessible through short-range initiation sites, such as those in anti-parallel sheets connected by turns. Before folding is completed, the system can encounter a second bottleneck, involving the condensation and rearrangement of surface residues. Overly stable local structure of the surface residues slows this stage of the folding process. The relation of the results from the 125-mer model studies to the folding of real proteins is discussed.     

Peroxynitrite-mediated nitration of tyrosine residues in Escherichia coli glutamine synthetase mimics adenylylation: relevance to signal transduction.

Treatment of Escherichia coli glutamine synthetase (GS) with peroxynitrite leads to nitration of some tyrosine residues and conversion of some methionine residues to methionine sulfoxide (MSOX) residues. Nitration, but not MSOX formation, is stimulated by Fe-EDTA. In the absence of Fe-EDTA, nitration of only one tyrosine residue per subunit of unadenylylated GS leads to changes in divalent cation requirement, pH-activity profile, affinity for ADP, and susceptibility to feedback inhibition by end products (tryptophan, AMP, CTP), whereas nitration of one tyrosine residue per subunit in the adenylylated GS leads to complete loss of catalytic activity. In the presence of Fe-EDTA, nitration is a more random process: nitration of five to six tyrosine residues per subunit is needed to convert unadenylylated GS to the adenylylated configuration. These results and the fact that nitration of tyrosine residues is an irreversible process serve notice that the regulatory function of proteins that undergo phosphorylation or adenylylation in signal transduction cascades might be seriously compromised by peroxynitrite-promoted nitration.     

Factor IX Cardiff: a variant factor IX protein that shows abnormal activation is caused by an arginine to cysteine substitution at position 145

Crude barium chloride eluates prepared from 12 unrelated patients with cross-reacting material positive (CRM+) haemophilia B were activated with celite eluate, the reaction products resolved after reduction by 13% SDS-PAGE, and factor IX antigenic material detected by probing with radiolabelled immunopurified rabbit anti-factor IX antiserum followed by autoradiography. Out of the 12, one sample showed faulty activation with the production of a stable reaction product with a MW compatible with that of a putative light chain-activation intermediate. In order to confirm this, two oligonucleotide primers that bracketed exon 6 of the factor IX gene were constructed and used to prime a polymerase chain reaction on DNA isolated from the patient's peripheral blood leucocytes. A single 489 nucleotide DNA fragment was obtained, gel purified, subcloned into M13, and DNA sequencing carried out on both strands. A single C to T transition was discovered that changed the Arg residue at position 145, the first residue of the first bond in the activation peptide, to a Cys, a result that confirmed the inferences drawn from the activation studies.     

Metabolic activation and detoxication of nephrotoxic cysteine and homocysteine S-conjugates.

S-(1,2-Dichlorovinyl)-L-homocysteine (DCVHcy), an analogue of the nephrotoxin S-(1,2-dichlorovinyl)-L-cysteine (DCVCys), is a much more potent nephrotoxin than DCVCys both in vivo and in isolated renal proximal tubular cells. S-(1,2-Dichlorovinyl)-DL-alpha-methylhomocysteine, at equimolar doses relative to DCVHcy, is not nephrotoxic. Agents that inhibit pyridoxal phosphate-dependent enzymes (DL-propargylglycine and aminooxyacetic acid) or renal organic anion transport (probenecid) protect against DCVHcy-induced nephrotoxicity. With kidney cytosol, DCVHcy or the analogue S-(2-benzothiazolyl)-L-homocysteine (BTHcy) is not metabolized to 2-ketobutyrate, but 2-mercaptobenzothiazole is a metabolite of BTHcy and the Vmax for its formation is enhanced by addition of 2-ketobutyrate. These results are consistent with a bioactivation mechanism for DCVHcy that involves enzymatic deamination followed by a nonenzymatic beta-elimination to produce two reactive intermediates--i.e., S-(1,2-dichlorovinyl)thiol and 2-keto-3-butenoic acid. The Km values for the N-acetylation of DCVCys and DCVHcy by kidney microsomal N-acetyltransferase are similar, but the rate of DCVCys N-acetylation is 4-fold greater than the rate measured with DCVHcy as the substrate. Thus, the remarkable nephrotoxic potency of DCVHcy compared with DCVCys may be attributable to intrarenal differences in activation and detoxication.