Structural requirements for thioester bond formation in human complement component C3. Reassessment of the role of thioester bond integrity on the conformation of C3.

A unique thioester bond, formed between the side chains of neighboring C and Q residues, is present in complement components C3 and C4 and the protease inhibitor alpha 2-macroglobulin. This structure is essential for mediating covalent attachment to target acceptors and also for maintaining these proteins in their native conformation. An examination of the residues in the immediate vicinity of the C and Q reveals a very high degree of sequence similarity among the three proteins which crosses species barriers. The following is the sequence flanking the thioester residues in C3, the highly conserved amino acids being underlined and the the thioester-forming residues being indicated by italics: 1005V-T-P-S-G-C-G-E-Q-N-M-I-G-M-T-P-T1021. Through a site-directed mutagenesis and cDNA expression approach, we have examined the importance of the conserved amino acids in the formation, stability, and function of the thioester bond in C3. The behavior of the mutants fell into three categories. The potential loss in peptide backbone flexibility by the replacement of G1009 by A or S was permissive to thioester formation and function as was replacement of M1015 by the still fairly bulky residue F. In contrast, replacement of M1015 by A resulted in an alpha-chain which was highly unstable toward proteolytic degradation. The third category, which included mutant molecules P1007G, P1020G, E1012Q, and Q1013N, displayed an unusual phenotype in which both the autolytic fragmentation and the hemolytic activity characteristics of thioester-intact molecules were absent. However, like their wildtype counterpart, these molecules retained the ability to be cleaved by C3 convertase (C4b2a), a conformation-dependent property that is normally lost in the conversion of native C3 to thioester-hydrolyzed C3(H2O). Since an identical functional profile was obtained when the thioester was deliberately prevented from forming in the mutant C1010A, we conclude that if a stable thioester fails to form during biosynthesis, at least parts of the mature protein can adopt a more native-like conformation than is the case when the thioester is first formed and then hydrolyzed in the mature protein. In view of these new findings, the interpretation of the previously observed correlation between the loss of thioester integrity and the adoption of a C3b-like conformation must be reassessed.     

Spontaneous thioester bond formation in alpha 2-macroglobulin, C3 and C4.

Purified alpha 2-macroglobulin and complement proteins C3 and C4 were treated with ammonia to break their intramolecular thioester bonds and reform the original free cysteinyl and glutamyl side chains. When this reaction was performed at low temperature a conformational intermediate was trapped which lacked a thioester, but which could refold to the native structure and spontaneously reform the thioester and full biological function. The findings suggest that these proteins may undergo spontaneous post-translational self-modification forming the thioesters without involvement of enzymes or high energy metabolites such as ATP.     

Mutation analysis of carbamoyl phosphate synthetase: Does the structurally conserved glutamine amidotransferase triad act as a functional dyad?

Evolutionarily conserved triad glutamine amidotransferase (GAT) domains catalyze the cleavage of glutamine to yield ammonia and sequester the ammonia in a tunnel until delivery to a variety of acceptor substrates in synthetase domains of variable structure. Whereas a conserved hydrolytic triad (Cys/His/Glu) is observed in the solved GAT structures, the specificity pocket for glutamine is not apparent, presumably because its formation is dependent on the conformational change that couples acceptor availability to a greatly increased rate of glutamine cleavage. In Escherichia coli carbamoyl phosphate synthetase (eCPS), one of the best characterized triad GAT members, the Cys269 and His353 triad residues are essential for glutamine hydrolysis, whereas Glu355 is not critical for eCPS activity. To further define the glutamine-binding pocket and possibly identify an alternative member of the catalytic triad that is situated for this role in the coupled conformation, we have analyzed mutations at Gln310, Asn311, Asp334, and Gln351, four conserved, but not yet analyzed residues that might potentially function as the third triad member. Alanine substitution of Gln351, Asn311, and Gln310 yielded respective K(m) increases of 145, 27, and 15, suggesting that Gln351 plays a key role in glutamine binding in the coupled conformation, and that Asn311 and Gln310 make less significant contributions. None of the mutant k (cat) values varied significantly from those for wild-type eCPS. Combined with previously reported data on other conserved eCPS residues, these results strongly suggest that Cys269 and His353 function as a catalytic dyad in the GAT site of eCPS.     

Identification of the cysteine residue exposed by the conformational change in pig heart succinyl-CoA:3-ketoacid coenzyme A transferase on binding coenzyme A

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) transfers CoA from succinyl-CoA to acetoacetate via a thioester intermediate with its active site glutamate residue, Glu 305. When CoA is linked to the enzyme, a cysteine residue can now be rapidly modified by 5,5'-dithiobis(2-nitrobenzoic acid), reflecting a conformational change of SCOT upon formation of the thioester. Since either Cys 28 or Cys 196 could be the target, each was mutated to Ser to distinguish between them. Like wild-type SCOT, the C196S mutant protein was modified rapidly in the presence of acyl-CoA substrates. In contrast, the C28S mutant protein was modified much more slowly under identical conditions, indicating that Cys 28 is the residue exposed on binding CoA. The specific activity of the C28S mutant protein was unexpectedly lower than that of wild-type SCOT. X-ray crystallography revealed that Ser adopts a different conformation than the native Cys. A chloride ion is bound to one of four active sites in the crystal structure of the C28S mutant protein, mimicking substrate, interacting with Lys 329, Asn 51, and Asn 52. On the basis of these results and the studies of the structurally similar CoA transferase from Escherichia coli, YdiF, bound to CoA, the conformational change in SCOT was deduced to be a domain rotation of 17 degrees coupled with movement of two loops: residues 321-329 that bury Cys 28 and interact with succinate or acetoacetate and residues 374-386 that interact with CoA. Modeling this conformational change has led to the proposal of a new mechanism for catalysis by SCOT.     

Mechanism of the beta-ketoacyl synthase reaction catalyzed by the animal fatty acid synthase

The catalytic mechanism of the beta-ketoacyl synthase domain of the multifunctional fatty acid synthase has been investigated by a combination of mutagenesis, active-site titration, product analysis, and product inhibition. Neither the reactivity of the active-site Cys161 residue toward iodoacetamide nor the rate of unidirectional transfer of acyl moieties to Cys161 was significantly decreased by replacement of any of the conserved residues, His293, His331, or Lys326, with Ala. Decarboxylation of malonyl moieties in the fully-active Cys161Gln background generated equimolar amounts of acetyl-CoA and bicarbonate, rather than carbon dioxide, and was seriously compromised by replacement of any of the conserved basic residues. The ability of bicarbonate to inhibit decarboxylation of malonyl moieties in the Cys161Gln background was significantly reduced by replacement of His293 but less so by replacement of His331. The data are consistent with a reaction mechanism, in which the initial primer transfer reaction is promoted largely through a lowering of the pKa of the Cys161 thiol by a helix dipole effect and activation of the substrate thioester carbon atom by binding of the keto group in an oxyanion hole. The data also indicate that an activated water molecule is present at the active site that is required either for the rapid hydration of carbon dioxide, prior its release as bicarbonate or, alternatively, for an initial attack on the malonyl C3. In the alternative mechanism, a negatively-charged tetrahedral transition state could be generated, stabilized in part by interaction of His293 with the negatively charged oxygen at C3 and interaction of His331 with the negatively charged thioester carbonyl oxygen, that breaks down to generate bicarbonate directly. Finally, the carbanion at C2, attacks the electrophilic C1 of the primer, generating a second tetrahedral transition state, also stabilized through contacts with the oxyanion hole and His331, that breaks down to form the beta-ketoacyl-S-acyl carrier protein product.     

Structural effects of cofilin on longitudinal contacts in F-actin

Structural effects of yeast cofilin on skeletal muscle and yeast actin were examined in solution. Cofilin binding to native actin was non-cooperative and saturated at a 1:1 molar ratio, with K(d)相似文献   

The conserved Cys76 plays a crucial role for the conformation of reduced glutathione peroxidase-type tryparedoxin peroxidase

The crystal structure of reduced tryparedoxin peroxidase shows Cys47 close to Gln82 and Trp137 and helix formation of residues 87 to 97 whereas the NMR structure of the reduced C76S mutant adopts a different conformation similar to the oxidized protein. Circular dichroism (CD), fluorescence and NMR spectroscopy reveal that the fully active C76S mutant differs from the wildtype (WT) enzyme mainly in its reduced form both in secondary structure content and Trp137 environment. This implies that Cys76 plays a critical role for the reduced enzyme assuming different conformational states and that the catalytic triad may only be necessary as short-lived intermediate during catalysis.     

Solution structure of the antimicrobial peptide gaegurin 4 by H and 15N nuclear magnetic resonance spectroscopy.

Gaegurin 4 (GGN4) is a 37-residue antimicrobial peptide isolated from the skin of a Korean frog, Rana rugosa. This peptide shows a broad range of activity against prokaryotic cells but shows very little hemolytic activity against human red blood cells. The solution structure of GGN4 was studied by using circular dichroism (CD) and NMR spectroscopy. CD investigations revealed that GGN4 adopts mainly an alpha-helical conformation in trifluoroethanol/water solution, in dodecylphosphocholine and in SDS micelles, but adopts random structure in aqueous solution. By using both homonuclear and heteronuclear NMR experiments, complete 1H and 15N resonance assignments were obtained for GGN4 in 50% trifluoroethanol/water solution. The calculated structures of GGN4 consist of two amphipathic alpha-helices extending from residues 2-10 and from residues 16-32. These two helices are connected by a flexible loop spanning between the residues 11 and 15. By using enzyme digestion and matrix-assisted laser desorption/ionization mass spectroscopy, we confirmed that GGN4 contains a disulfide bridge formed between the residues Cys31 and Cys37 in its C-terminus. The effect of disulfide bridge on the structure and the activity of GGN4 was investigated. The reduced form of GGN4 revealed a similar activity and conformation to native GGN4, suggesting that the disulfide bridge does not strongly affect the conformation and the antimicrobial activity of GGN4.     

A study of renaturation of reduced hen egg white lysozyme. Enzymically active intermediates formed during oxidation of the reduced protein.

The material obtained from reduced hen egg white lysozyme after complete air oxidation at pH 8.0 and 37 degrees has yielded, by gel filtration on a Bio-Gel P-30 column, enzymically active species and an enzymically inactive form which eluted sooner than the active species but later than expected for a dimer of lysozyme. Reduced lysozyme also elutes at the same position as this inactive material. Examination of the fragments produced on CNBr cleavage of the inactive form indicates that at least 24% of the population contains incorrect disulfide bonds involving half-cystine residues 6, 30, 115, and 127. Tryptophan fluorescence and the intrinsic viscosity of the inactive form show an enlarged molecular domain with a disordered conformation. The yield of the inactive form increases as the oxidation of reduced lysozyme is accelerated using cupric ion. In the presence of 4 X 10(-5) M cupric ion, reduced lysozyme forms almost quantitatively the inactive form, which is almost completely converted to the native form by sulfhydryl-disulfide interchange catalyzed by thiol groups of either reduced lysozyme or beta-mercaptoethanol. The material trapped by alkylation of the free sulfhydryl groups with [1-14C]iodoacetic acid during the early stage of air oxidation of reduced lysozyme was fractionated by gel filtration to permit separation of the active species from the inactive form. Ion exchange chromatography of the active species yielded completely renatured lysozyme and three major enzymically active radioactive derivatives. Two of these derivatives contained approximately 2 mol of S-carboxymethylcysteine. Isolation and characterization of radioactive tryptic peptides from each of the three active forms, permitted the identification of Cys 6 and Cys 127, Cys 76 and 94, and Cys 80 as the sulfhydryl groups alkylated in these three incompletely oxidized, partially active forms. Thus, it appears that the interatomic interactions maintaining the compact three-dimensional structure of native lysozyme are operational even when one of these three native disulfide bonds between Cys 6 and Cys 127, Cys 76 and Cys 94, and Cys 64 and 80 is open.     

The mitochondrial citrate transport protein: evidence for a steric interaction between glutamine 182 and leucine 120 and its relationship to the substrate translocation pathway and identification of other mechanistically essential residues

Previous examination of the accessibility of a panel of single-Cys mutants in transmembrane domain III (TMDIII) of the yeast mitochondrial citrate transport protein to the hydrophilic, cysteine-specific methanethiosulfonate reagent MTSES enabled identification of the water-accessible surface of this TMD. Further studies on the effect of citrate on MTS reagent accessibility, indicated eight sites within TMD III at which citrate conferred temperature-independent protection, thus providing strong evidence for participation of these residues in the formation of a portion of the substrate translocation pathway. Unexpectedly, citrate did not protect against inhibition of the Leu120Cys variant, despite its location on a water- and citrate-accessible surface of the TMDIII helix. This led to the hypothesis that in the 3-dimensional CTP structure, TMDIV packs against TMDIII in a manner such that the Leu120 side-chain folds behind the side-chain of Gln182. The present investigations addressed this hypothesis by examining the properties of the Gln182Cys single mutant and the Leu120Cys/Gln182Ala double mutant. We observed that in contrast to our findings with the Leu120Cys mutant, citrate did protect the Gln182Cys variant against MTSES-mediated inhibition. Importantly, truncation of the Gln182 side-chain to Ala enabled citrate to protect the Leu120Cys double mutant against inhibition. In combination these data support the idea that the Gln182 side-chain lines the transport path and sterically blocks access of citrate to the Leu120 side-chain. In a parallel series of investigations, we constructed 24 single-Cys substitution mutants that were chosen based on their hypothesized importance in substrate binding and/or translocation. We observed that substitution of Cys for residues E34, K37, K83, R87, Y148, D236, K239, T240, R276, and R279 resulted in > or =98% inactivation of CTP function, suggesting an essential structural and/or mechanistic role for these native residues. Superposition of this functional data onto a detailed 3-dimensional homology model of the CTP structure indicates that the side-chains of each of these residues project into the putative transport pathway. We hypothesize that a subset of these residues, in combination with four previously identified essential residues, define the citrate binding site(s) within the CTP.     

Two-dimensional 1H nuclear magnetic resonance study of the (5-55) single-disulphide folding intermediate of bovine pancreatic trypsin inhibitor

An analogue of the bovine pancreatic trypsin inhibitor (BPTI) folding intermediate that contains only the disulphide bond between Cys5 and Cys55 has been prepared in Escherichia coli by protein engineering methods, with the other four Cys residues replaced by Ser. Two-dimensional 1H nuclear magnetic resonance studies of the analogue have resulted in essentially complete resonance assignments of the folded form of the protein. The folded protein has a compact conformation that is structurally very similar to that of native BPTI, although there are subtle differences and the folded conformation is not very stable. Approximately half of the protein molecules are unfolded at 3 degrees C, and this proportion increases at higher temperatures. The folded and unfolded conformations are in slow exchange. The conformational properties of the analogue can explain many aspects of the kinetic role that the normal (5-55) intermediate plays in the folding of BPTI.     

Conformation of NAD+ bound to allosteric L-lactate dehydrogenase activated by chemical modification

On modification of arginine residues with 2,3-butanedione, the Thermus caldophilus L-lactate dehydrogenase is converted to an activated form that is independent of an allosteric effector, fructose 1,6-bisphosphate (Fru-1,6-P2). The conformation of NAD+ bound to the modified enzyme in the absence of Fru-1,6-P2 was investigated by means of proton NMR, analyzing the time dependence of the transferred nuclear Overhauser effect (TRNOE) and TRNOE action spectra. The inter-proton distances determined on TRNOE analysis indicated that both the nicotinamide riboside moiety and the adenosine moiety of NAD+ were in the anti conformation, the ribose rings being in the C3'-endo form. This conformation was almost the same as that of NAD+ bound to the native enzyme-Fru-1,6-P2 complex, rather than that of NAD+ bound to the free native enzyme. These results suggest that the C3'-endo-anti form of the enzyme-bound NAD+ is essential for the activation of the T. caldophilus L-lactate dehydrogenase.     

Human liver manganese superoxide dismutase. Purification and crystallization, subunit association and sulfhydryl reactivity

Manganese superoxide dismutase (Mn-SOD) has been purified with a high yield (320 mg) from human liver (2 kg) and crystallized. Low-angle laser light scattering of the enzyme has shown that native enzyme is a tetrametic form. Four of the eight cysteine residues in the tetramer reacted with 5,5'-dithiobis(2-nitrobenzoic acid) or with iodoacetamide. The others were only reactive in protein heated with SDS or urea after reduction with dithiothreitol or 2-mercaptoethanol. The reactive sulfhydryl group was found to be located at Cys196 by amino acid sequence analysis of Nbs2-reactive peptides isolated by activated thiol-Sepharose covalent chromatography. Incubation of Mn-SOD in 1% SDS for 2 or 3 days at 25 degrees C or 5 min at 100 degrees C gave material showing two prominent components on polyacrylamide gel electrophoresis in the presence of 0.1% SDS. The major component had a molecular mass of 23 kDa; the other, 25 kDa. Reduction of the protein by dithiothreitol or 2-mercaptoethanol heated in SDS produced only the 25-kDa monomer species. Essentially, no thiol groups were detected in the 23-kDa form, in which two cysteine residues appear to have been oxidized to form an intrasubunit disulfide. This indicates that Cys196 has a reactive sulfhydryl and appears to be a likely candidate for a mixed disulfide formation in vivo.     

Functional fine-mapping and molecular modeling of a conserved loop epitope of the measles virus hemagglutinin protein.

Neutralizing and protective monoclonal antibodies (mAbs) were used to fine-map the highly conserved hemagglutinin noose epitope (H379-410, HNE) of the measles virus. Short peptides mimicking this epitope were previously shown to induce virus-neutralizing antibodies [El Kasmi et al. (2000) J. Gen. Virol.81, 729-735]. The epitope contains three cysteine residues, two of which (Cys386 and Cys394) form a disulfide bridge critical for antibody binding. Substitution and truncation analogues revealed four residues critical for binding (Lys387, Gly388, Gln391 and Glu395) and suggested the binding motif X7C[KR]GX[AINQ]QX2CEX5 for three distinct protective mAbs. This motif was found in more than 90% of the wild-type viruses. An independent molecular model of the core epitope predicted an amphiphilic loop displaying a remarkably stable and rigid loop conformation. The three hydrophilic contact residues Lys387, Gln391 and Glu395 pointed on the virus towards the solvent-exposed side of the planar loop and the permissive hydrophobic residues Ile390, Ala392 and Leu393 towards the solvent-hidden side of the loop, precluding antibody binding. The high affinity (Kd = 7.60 nm) of the mAb BH216 for the peptide suggests a high structural resemblance of the peptide with the natural epitope and indicates that most interactions with the protein are also contributed by the peptide. Improved peptides designed on the basis of these findings induced sera that crossreacted with the native measles virus hemagglutinin protein, providing important information about a lead structure for the design of more stable antigens of a synthetic or recombinant subunit vaccine.     

Evidence for the proximity of the extreme N-terminus of the neurokinin-2 (NK2) tachykinin receptor to Cys167 in the putative fourth transmembrane helix

Neurokinin-2 receptor (NK(2)R) binding of [(3)H]-SR48968, a piperidinyl antagonist, is inhibited by methanethiosulfonate ethylammonium (MTSEA) in a time- and concentration-dependent manner. By the systematic alanine replacement of putative loop and transmembrane region cysteine residues (Cys(4), Cys(81), Cys(167), Cys(262), Cys(281), Cys(308), and Cys(309)), we have determined that MTSEA perturbs [(3)H]-SR48968 binding by modifying Cys(167) in transmembrane helix 4. Data were substantiated using glycine, serine, and threonine substitutions of Cys(167). MTSEA preferentially modifies cysteine residues that are in proximity to a negatively charged environment. Hence, aspartate and glutamate residues were systematically substituted with leucine or valine, respectively, and the inhibitory effects of MTSEA on [(3)H]-SR48968 binding were reevaluated to determine those acidic residues close to the MTSEA binding crevice. Most significantly, substitution of Asp(5) in the receptor's extreme N-terminus abolished the effects of MTSEA on [(3)H]-SR48968 binding. Therefore, our data would suggest close association of the extreme N-terminus with the extracellular surfaces of helices 4 and 3 in the NK(2)R in forming a binding crevice for MTSEA. The inhibition of SR48968 binding appears to result from loss of the SR48968 binding conformation of Gln(166) induced by MTSEA when it is coupled to Cys(167). Hence, it is proposed that there is mutually exclusive hydrogen bonding of SR48968 and MTSEA to Gln(166).     

The structure around the thioester bond in bovine alpha 2-macroglobulin. Possible implications for the conformational stability of the inhibitor on thioester cleavage

The residues contributing to the thioester bonds in bovine alpha 2-macroglobulin were differentially labelled by modification of the Glu moiety with [14C]methylamine and of the Cys moiety with iodo[3H]acetate. The labelled region was identified and analyzed in a tryptic peptide. Two amino acid replacements between human and bovine alpha 2-macroglobulin were found at positions +3 (Val/Ala) and +4 (Leu/Arg) from the Glu moiety of the thioester. Thus, marked differences exist between the human and bovine proteins in side chain size and charge close to the thioester bonds. These differences may explain the greater conformational stability of bovine alpha 2-macroglobulin, compared with that of the human inhibitor, after cleavage of the thioester bonds.     

Reactivities of the S2 and S3 subsite residues of thrombin with the native and heparin-induced conformers of antithrombin.

A pentasaccharide (PS) fragment of heparin capable of activating antithrombin (AT) markedly accelerates the inhibition of factor Xa by AT, but has insignificant effect on inhibition of thrombin. For inhibition of thrombin, the bridging function of a longer polysaccharide chain is required to accelerate the reaction. To study the basis for the similar reactivity of thrombin with the native or heparin-activated conformers of AT, several residues surrounding the active site pocket of thrombin were targeted for mutagenesis study. Leu99 and Glu192, the variant residues influencing the S2 and S3 subsite specificity of thrombin were replaced with Tyr and Gln. The Tyr60a, Pro60b, Pro60c, and Trp60d residues forming part of the S2 specificity pocket were deleted from the B-insertion loop of the wild-type and Leu99/Glu192 --> Tyr/Gln thrombins. Kinetic studies indicated that the reactivities of all mutants with AT were moderately or severely impaired. Although heparin largely corrected the defect in reactivities, it also markedly elevated the stoichiometries of inhibition with the mutants. Interestingly, PS also accelerated AT inhibition of the mutants 5-68-fold, suggesting that the mutants are able to discriminate between the native and activated conformers of AT. Based on these results and the recent crystal structure determination of AT in complex with PS, a model for thrombin-AT interaction is proposed in which the S2 and S3 subsite residues of thrombin are critical for recognition of the P2 and P3 residues of AT in the native conformation. In the activated conformation, other residues are made accessible for interaction with the protease, and the similar reactivity of thrombin with the native and heparin-activated conformers of AT may be coincidental. The results further suggest that the S2 and S3 subsite residues are crucial in controlling the partitioning of the thrombin-AT intermediate into the alternative inhibitory or substrate pathways of the reaction.     

Use of time-resolved FRET to validate crystal structure of complement regulatory complex between C3b and factor H (N terminus)

Structural knowledge of interactions amongst the ～ 40 proteins of the human complement system, which is central to immune surveillance and homeostasis, is expanding due primarily to X‐ray diffraction of co‐crystallized proteins. Orthogonal evidence, in solution, for the physiological relevance of such co‐crystal structures is valuable since intermolecular affinities are generally weak‐to‐medium and inter‐domain mobility may be important. In this current work, Förster resonance energy transfer (FRET) was used to investigate the 10 μM K_D (210 kD) complex between the N‐terminal region of the soluble complement regulator, factor H (FH1‐4), and the key activation‐specific complement fragment, C3b. Using site‐directed mutagenesis, seven cysteines were introduced individually at potentially informative positions within the four CCP modules comprising FH1‐4, then used for fluorophore attachment. C3b possesses a thioester domain featuring an internal cycloglutamyl cysteine thioester; upon hydrolysis this yields a free thiol (Cys988) that was also fluorescently tagged. Labeled proteins were functionally active as cofactors for cleavage of C3b to iC3b except for FH1‐4(Q40C) where conjugation with the fluorophore likely abrogated interaction with the protease, factor I. Time‐resolved FRET measurements were undertaken to explore interactions between FH1‐4 and C3b in fluid phase and under near‐physiological conditions. These experiments confirmed that, as in the cocrystal structure, FH1‐4 binds to C3b with CCP module 1 furthest from, and CCP module 4 closest to, the thioester domain, placing subsequent modules of FH near to any surface to which C3b is attached. The data do not rule out flexibility of the thioester domain relative to the remainder of the complex.     

Kinetic analysis of the reactions of hypobromous acid with protein components: implications for cellular damage and use of 3-bromotyrosine as a marker of oxidative stress

Hypohalous acids (HOX, X = Cl, Br) are produced by activated neutrophils, monocytes, eosinophils, and possibly macrophages. These oxidants react readily with biological molecules, with amino acids and proteins being major targets. Elevated levels of halogenated Tyr residues have been detected in proteins isolated from patients with atherosclerosis, asthma, and cystic fibrosis, implicating the production of HOX in these diseases. The quantitative significance of these findings requires knowledge of the kinetics of reaction of HOX with protein targets, and such data have not been previously available for HOBr. In this study, rate constants for reaction of HOBr with protein components have been determined. The second-order rate constants (22 degrees C, pH 7.4) for reaction with protein sites vary by 8 orders of magnitude and decrease in the order Cys > Trp approximately Met approximately His approximately alpha-amino > disulfide > Lys approximately Tyr > Arg > backbone amides > Gln/Asn. For most residues HOBr reacts 30-100 fold faster than HOCl, though Cys and Met residues are approximately 10-fold less reactive, and ring halogenation of Tyr is approximately 5000-fold faster. Thus, Tyr residues are more, and Cys and Met much less, important targets for HOBr than HOCl. Kinetic models have been developed to predict the targets of HOX attack on proteins and free amino acids. Overall, these results shed light on the mechanisms of cell damage induced by HOX and indicate, for example, that the 3-chloro-Tyr:3-bromo-Tyr ratio does not reflect the relative roles of HOCl and HOBr in disease processes.