Disulfide bridges in tomato pectinesterase: variations from pectinesterases of other species; conservation of possible active site segments.

Analysis of tomato pectinesterase by carboxymethylation, with and without reduction, shows that the enzyme has two intrachain disulfide bridges. Analysis of fragments obtained from the native enzyme after digestion with pepsin identified bridges connecting Cys-98 with Cys-125, and Cys-166 with Cys-200. The locations of disulfide bridges in tomato pectinesterase are not identical to those in three distantly related pectinesterases (18-33% residue identities) from microorganisms. However, one half-Cys (i.e., Cys-166) position is conserved in all four enzymes. Sequence comparisons of the overall structures suggest a special importance for three short segments of the entire protein. One segment is at the N-terminal part of the tomato pectinesterase, another in the C-terminal portion near the distal end of the second disulfide loop, and the third segment is located in the central part between the two disulfide bridges. The latter segment, encompassing only 40 residues of the entire protein, appears to high-light a functional site in a midchain segment.     

The amino acid sequence of the sex steroid-binding protein of rabbit serum

The amino acid sequence of the sex steroid-binding protein (SBP or SHBG) of rabbit serum, specific for binding testosterone and 5 alpha-dihydrotestosterone, was determined using a complementary combination of mass spectrometric and Edman degradation techniques. The monomeric unit of the homodimeric protein is a single chain glycopeptide of 367 amino acid residues, with N-linked oligosaccharide side chains at Asn-345 and Asn-361 and disulfide bonds connecting Cys-158 to Cys-182 and Cys-327 to Cys-355. The polypeptide molecular weight of the monomer calculated from the sequence is 39,769. The molecular weight of the homodimer including 9% carbohydrate is 87,404. The sequence contains a relatively hydrophobic segment between Trp-241 and Leu-282, which includes many leucine residues in an alternating pattern. An amino acid sequence repeat is also located within that segment. Both of these patterns are present in human SBP and in the androgen-binding protein of rat epididymis. The sequence data indicate that the previously reported microheterogeneity of rabbit SBP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis reflects variants generated by differential glycosylation of the monomer rather than different gene products. Seventy-nine percent of the amino acids of rabbit SBP are identical to those of human SBP; rabbit SBP thus joins human SBP and rat androgen-binding protein in one gene family that is distinct from the steroid hormone receptor superfamily. It appears that the problem of binding sex steroid hormones has been solved independently in two different gene families that contain completely different steroid-binding domains. Since the nonhomologous steroid-binding domains of both families of proteins recognize essentially the same steroid structure, it will be interesting to determine the structural basis of the two different protein designs that lead to similar steroid-binding specificity.     

Primary structure of hemorrhagic protein, HR2a, isolated from the venom of Trimeresurus flavoviridis

The complete amino acid sequence and disulfide bridge location of HR2a, one of the hemorrhagic proteins isolated from the snake venom of Trimeresurus flavoviridis, have been determined by analysis of peptides derived from digests with cyanogen bromide, lysyl endopeptidase, trypsin, and Staphylococcus aureus V8 protease. Peptides were purified by gel filtration followed by reversed-phase HPLC. HR2a has the amino-terminal sequence of less than Glu-Gln-Arg- and consists of a total of 202 residues with a calculated molecular weight of 23,015. Sequence analysis indicates the presence of another isoform which lacks the amino-terminal residue, making 201 amino acid residues with a molecular weight of 22,887. Three disulfide bridges of HR2a link Cys-118 to Cys-197, Cys-159 to Cys-181, and Cys-161 to Cys-164. HR2a contains a segment which is similar to the zinc-chelating sequences found in thermolysin and several mammalian metalloproteinases, suggesting that HR2a is a metalloproteinase with limited substrate specificity. However, there is no other significant sequence homology with thermolysin except for the zinc-ligand region.     

Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry

Hydrogen/deuterium (H/D) exchange in combination with electrospray ionization mass spectrometry and near-ultraviolet (UV) circular dichroism (CD) was used to study the conformational properties and thermal unfolding of Escherichia coli thioredoxin and its Cys32-alkylated derivatives in 1% acetic acid (pH 2.7). Thermal unfolding of oxidized (Oxi) and reduced (Red) -thioredoxin (TRX) and Cys-32-ethylglutathionyl (GS-ethyl-TRX) and Cys-32-ethylcysteinyl (Cys-ethyl-TRX), which are derivatives of Red-TRX, follow apparent EX1 kinetics as charge-state envelopes, H/D mass spectral exchange profiles, and near-UV CD appear to support a two-state folding/unfolding model. Minor mass peaks in the H/D exchange profiles and nonsuperimposable MS- and CD-derived melting curves, however, suggest the participation of unfolding intermediates leading to the conclusion that the two-state model is an oversimplification of the process. The relative stabilities as measured by melting temperatures by both CD and mass spectral charge states are, Oxi-TRX, GS-ethyl-TRX, Cys-ethyl-TRX, and Red-TRX. The introduction of the Cys-32-ethylglutathionyl group provides extra stabilization that results from additional hydrogen bonding interactions between the ethylglutathionyl group and the protein. Near-UV CD data show that the local environment near the active site is perturbed to almost an identical degree regardless of whether alkylation at Cys-32 is by the ethylglutathionyl group, or the smaller, nonhydrogen-bonding ethylcysteinyl group. Mass spectral data, however, indicate a tighter structure for GS-ethyl-TRX.     

Studies on the structure of rabbit muscle aldolase: Ordering of the tryptic peptides; Sequence of 164 amino acid residues in the NH2-terminal BrCN peptide

The sequence of 164 amino acid residues in the NH₂-terminal BrCN peptide of rabbit muscle aldolase has been determined. The information has permitted location of the following amino acid residues involved in the catalytic activity or in maintaining the structural integrity of the enzyme: Cys-72, forms a disulfide bridge with Cys-336 in the COOH-terminal segment on inactivation of the enzyme by oxidation; Lys-107, forms a Schiff base with pyridoxal phosphate upon inactivation of aldolase by this reagent; Cys-134 and Cys-177, buried, do not react with SH-reagents in the native enzyme.     

A conformationally sensitive residue on the cytoplasmic surface of serotonin transporter

Serotonin transporter (SERT) contains a single reactive external cysteine residue at position 109 (Chen, J. G., Liu-Chen, S., and Rudnick, G. (1997) Biochemistry 36, 1479-1486) and seven predicted cytoplasmic cysteines. A mutant of rat SERT (X8C) in which those eight cysteine residues were replaced by other amino acids retained approximately 32% of wild type transport activity and approximately 56% of wild type binding activity. In contrast to wild-type SERT or the C109A mutant, X8C was resistant to inhibition of high affinity cocaine analog binding by the cysteine reagent 2-(aminoethyl)methanethiosulfonate hydrobromide (MTSEA) in membrane preparations from transfected cells. Each predicted cytoplasmic cysteine residue was reintroduced, one at a time, into the X8C template. Reintroduction of Cys-357, located in the third intracellular loop, restored MTSEA sensitivity similar to that of C109A. Replacement of only Cys-109 and Cys-357 was sufficient to prevent MTSEA sensitivity. Thus, Cys-357 was the sole cytoplasmic determinant of MTSEA sensitivity in SERT. Both serotonin and cocaine protected SERT from inactivation by MTSEA at Cys-357. This protection was apparently mediated through a conformational change following ligand binding. Although both ligands bind in the absence of Na(+) and at 4 degrees C, their ability to protect Cys-357 required Na(+) and was prevented at 4 degrees C. The accessibility of Cys-357 to MTSEA inactivation was increased by monovalent cations. The K(+) ion, which is believed to serve as a countertransport substrate for SERT, was the most effective ion for increasing Cys-357 reactivity.     

Locations of the six disulphide bonds in a variant surface glycoprotein (VSG 117) from Trypanosoma brucei.

The locations of the six disulphide bonds and the single free cysteine residue in a variant surface glycoprotein, VSG 117, from the African trypanosome Trypanosoma brucei have been determined to be Cys-14--Cys-140, Cys-121--Cys-182, Cys-389--Cys-404, Cys-398--417, Cys-447--Cys-461 and Cys-455--Cys-468. Cys-244 bears the single thiol group, which is unreactive towards 2-nitro-5-thiocyanobenzoate in the native molecule and is probably buried. Biosynthetically incorporated [35S]cysteine aided the location of the disulphide bonds. Two proteinase-resistant glycosylated domains, each containing two disulphide bonds, were identified in the C-terminal region of the glycoprotein. Details of purification of [35S]cysteine-containing peptides, and Tables of amino acid analyses, are presented in Supplementary Publication SUP 50119 (32 pages), which has been deposited with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1981) 193,5.     

MgADP-induced changes in the structure of myosin S1 near the ATPase-related thiol SH1 probed by cross-linking

The structural consequences of MgADP binding at the vicinity of the ATPase-related thiol SH1 (Cys-707) have been examined by subjecting myosin subfragment 1, premodified at SH2 (Cys-697) with N-ethylmaleimide (NEM), to reaction with the bifunctional reagent p-phenylenedimaleimide (pPDM) in the presence and absence of MgADP. By monitoring the changes in the Ca2(+)-ATPase activity as a function of reaction time, it appears that the reagent rapidly modifies SH1 irrespective of whether MgADP is present or not. In the absence of nucleotide, only extremely low levels of cross-linking to the 50-kDa middle segment of S1 can be detected, while in the presence of MgADP substantial cross-linking to this segment is observed. A similar cross-link is also formed if MgADP is added subsequent to the reaction of the SH2-NEM-pre-modified S1 with pPDM in the absence of nucleotide. Isolation of the labeled tryptic peptide from the cross-linked adduct formed with [14C]pPDM, and subsequent partial sequence analyses, indicates that the cross-link is made from SH1 to Cys-522. Moreover, it appears that this cross-link results in the trapping of MgADP in this S1 species. These data suggest that the binding of MgADP results in a change in the structure of S1 in the vicinity of the SH1 thiol relative to the 50-kDa "domain" which enables Cys-522 to adopt the appropriate configuration to enable it to be cross-linked to SH1 by pPDM.     

Assembly of the bacteriophage T4 helicase: architecture and stoichiometry of the gp41-gp59 complex

The bacteriophage T4 59 protein (gp59) plays an essential role in recombination and replication by mediating the assembly of the gene 41 helicase (gp41) onto DNA. gp59 is required to displace the gp32 single-stranded binding protein on the lagging strand to expose a site for helicase binding. To gain a better understanding of the mechanism of helicase assembly, the architecture and stoichiometry of the gp41-gp59 complex were investigated. Both the N and C termini of gp41 were found to lie close to or in the gp41-gp41 subunit interface and interact with gp59. The site of interaction of gp41 on gp59 is proximal to Cys-215 of gp59. Binding of gp41 to gp59 stimulates a conformational change in the protein resulting in hexamer formation of gp59, and gp59 likewise stimulates oligomer formation of gp41. The gp59 subunits in this complex are arranged in a head to head orientation, such that Cys-42 of one subunit is in close proximity to Cys-42 on an adjacent subunit, and Cys-215 on one subunit is close to Cys-215 on a neighboring subunit. As the helicase is loaded onto DNA, a conformational change in the gp41-gp59 complex occurs, which may serve to displace gp32 from the lagging strand and load the hexameric helicase in its place.     

Design and synthesis of the pseudo-EF hand in calbindin D9K: effect of amino acid substitutions in the alpha-helical regions

A series of 37-residue analogues of the pseudo-EF hand in bovine calbindin D9K has been synthesized by the solid phase method. In the presence of calcium an alpha-helical induction of up to 44% was observed for the peptide with the native sequence with a Kd for calcium binding of 0.35 mM. A number of amino acid substitutions have been carried out to study the packing of the two alpha-helices based on the crystal structure of the entire protein. Three strategies were employed: (1) replacement of the Leu residues, which in the crystal structure do not contribute to the hydrophobic interaction between the two helices, by Gln or Ala in order to control the orientation of the helix packing, (2) stabilization of the individual helix by introducing a Glu-...Lys+ salt bridge or by changing the N-terminal charge to compensate for the helix dipole moment, and (3) introduction of a disulfide bond between the two helices to help the packing of the helices. The mutants with the substitution of (Leu-30, Leu-32) to (Gln-30, Gln-32), (Gln-30, Ala-32), and (Ala-30,Ala-32) designed based on the strategy 1 do not show any affinity for calcium and have low alpha-helicity. The Leu-30 to Lys-30 mutant designed to form a salt bridge between the side chains of Glu-26 and Lys-30 has an apparent Kd for calcium of 6.8 mM. Kd of the N-terminal acetylated and succinylated mutants are 0.41 and 0.45 mM, respectively, and no increase in the alpha-helix content relative to that of the natural sequence peptide is observed. The disulfide containing mutants, namely Tyr-13, Leu-31 to Cys-13, Cys-31 and Tyr-13, Leu-31 to Cys-13, hCys-31, show apparent Kd values of 0.93 and 2.1 mM, respectively. The former mutant shows the highest alpha-helix content among the peptides studied in the presence and absence of calcium. While it is difficult to construct an isolated and rigid helix-loop-helix motif with peptides of this size, introduction of a disulfide bond proved to be effective for this purpose.     

The uptake inhibitors cocaine and benztropine differentially alter the conformation of the human dopamine transporter

The binding affinity of the cocaine analog [(3)H]2 beta-carbomethoxy-3beta-(4-fluorophenyl) tropane (WIN) for the dopamine transporter (DAT) is increased by the reaction of Cys-90, at the extracellular end of the first transmembrane segment, with methanethiosulfonate (MTS) reagents. Cocaine enhances the reaction of Cys-90 with the sulfhydryl reagents, thereby augmenting the increase in binding. In contrast, cocaine decreases the reaction of Cys-135 and Cys-342, endogenous cysteines in cytoplasmic loops, with MTS reagents. Because this reaction inhibits [(3)H]WIN binding, cocaine protects against the loss of binding caused by reaction of these cysteines. In the present work, we compare the abilities of DAT inhibitors and substrates to affect the reaction of Cys-90, Cys-135, and Cys-342 with MTS ethyltrimethylammonium (MTSET). The results indicate that the different abilities of compounds to protect against the MTSET-induced inhibition of binding are attributable to differences in their abilities to attenuate the inhibitory effects of modification of Cys-135 and Cys-342 as well as to enhance the reaction with Cys-90 and the resulting potentiation of binding. The inhibitor benztropine was unique in its inability to protect Cys-135. Moreover, whereas cocaine, WIN, mazindol, and dopamine enhanced the reaction of Cys-90 with MTSET, benztropine had no effect on this reaction. These two features combine to give benztropine its weak potency in protecting ligand binding to wild-type DAT from MTSET. These results indicate that different inhibitors of DAT, such as cocaine and benztropine, produce different conformational changes in the transporter. There are differences in the psychomotor stimulant-like effects of these compounds, and it is possible that the different behavioral effects of these DAT inhibitors stem from their different molecular actions on DAT.     

Deciphering the role of histidine 252 in mycobacterial adenosine 5'-phosphosulfate (APS) reductase catalysis

Mycobacterium tuberculosis adenosine 5'-phosphosulfate reductase (APR) catalyzes the first committed step in sulfate reduction for the biosynthesis of cysteine and is essential for survival in the latent phase of tuberculosis infection. The reaction catalyzed by APR involves the nucleophilic attack by conserved Cys-249 on adenosine 5'-phosphosulfate, resulting in a covalent S-sulfocysteine intermediate that is reduced in subsequent steps by thioredoxin to yield the sulfite product. Cys-249 resides on a mobile active site lid at the C terminus, within a K(R/T)ECG(L/I)H motif. Owing to its strict conservation among sulfonucleotide reductases and its proximity to the active site cysteine, it has been suggested that His-252 plays a key role in APR catalysis, specifically as a general base to deprotonate Cys-249. Using site-directed mutagenesis, we have changed His-252 to an alanine residue and analyzed the effect of this mutation on the kinetic parameters, pH rate profile, and ionization of Cys-249 of APR. Interestingly, our data demonstrate that His-252 does not perturb the pK(a) of Cys-249 or play a direct role in rate-limiting chemical steps of the reaction. Rather, we show that His-252 enhances substrate affinity via interaction with the α-phosphate and the endocyclic ribose oxygen. These findings were further supported by isothermal titration calorimetry to provide a thermodynamic profile of ligand-protein interactions. From an applied standpoint, our study suggests that small-molecules targeting residues in the dynamic C-terminal segment, particularly His-252, may lead to inhibitors with improved binding affinity.     

Acid-base catalysis in the mechanism of thioredoxin reductase from Drosophila melanogaster

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin (Trx) by NADPH. Like other members of the pyridine nucleotide-disulfide oxidoreductase enzyme family, the enzyme from Drosophila melanogaster is a homodimer, and each catalytically active unit consists of three redox centers: FAD and an N-terminal Cys-57/Cys-62 redox-active disulfide from one monomer and a Cys-489'/Cys-490' C-terminal redox-active disulfide from the second monomer. Because dipteran insects such as D. melanogaster lack glutathione reductase, thioredoxin reductase (DmTrxR) is particularly important; in addition to its normal functions, it also reduces GSSG for antioxidant protection. DmTrxR, used as a model for the enzyme from the malaria vector, Anopheles gambiae, has been shown to cycle in catalysis between the two-electron and four-electron reduced states, EH2 and EH4 [Bauer, H. et al. (2003) J. Biol. Chem. 278, 33020-33028]. His-464' acts as an acid-base catalyst of the dithiol-disulfide interchange reactions required in catalysis. The H464'Q enzyme has only 2% of the wild-type activity, emphasizing the importance of this residue. The pH dependence of Vmax for wild-type DmTrxR has pKa values of 6.4 and 9.3 on the DmTrxR-DmTrx-2 complex, whereas H464'Q DmTrxR only has an observable pKa at 6.4, indicating that the pKa at pH 9.3 is contributed mainly by His-464'. The pKa at pH 6.4 has been assigned to Cys-57 and Cys-490'; the thiolate on Cys-490' is the nucleophile in the reduction of Trx. In contrast to wild-type DmTrxR, H464'Q DmTrxR does not stabilize a thiolate-FAD charge-transfer complex in the presence of excess NADPH. The rates of steps in both the reductive and the oxidative half-reactions are markedly diminished in H464'Q DmTrxR as compared to those of wild-type enzyme, indicating that His-464' is involved in both half-reactions.     

Secondary structure and hydrogen bonding of crambin in solution. A two-dimensional NMR study

The secondary structure of crambin in solution has been determined using two-dimensional NMR and is found to be essentially identical to that of the crystal structure. The H-D exchange of most amide protons can be accounted for in terms of the hydrogen bonds found in the X-ray structure. Exceptions are the amide protons of Cys-4 and Ser-6, which exchange more slowly than expected, and of Asn-46 for which the exchange is faster. These results might be explained by a slightly different conformation of the C-terminal region of the protein in solution. The slow exchange of the amides of Cys-32 and Glu-23 might be due to aggregation involving an extremely hydrophobic part of the protein in solution.     

Evidence for a new sub-class of methionine sulfoxide reductases B with an alternative thioredoxin recognition signature

Methionine sulfoxide reductases catalyze the reduction of protein-bound methionine sulfoxide back to methionine via a thioredoxin-recycling process. Two classes of methionine sulfoxide reductases, called MsrA and MsrB, exist that display opposite stereoselectivities toward the sulfoxide function. Although they are structurally unrelated, they share a similar chemical mechanism that includes three steps with 1) formation of a sulfenic acid intermediate with a concomitant release of 1 mol of methionine per mole of enzyme; 2) formation of an intradisulfide Msr bond; and 3) reduction of the oxidized Msr by thioredoxin. In the MsrBs that have been biochemically, enzymatically, and structurally characterized so far, the cysteine involved in the regeneration of the catalytic Cys-117 is Cys-63. Cys-117 is located on a beta strand, whereas the recycling Cys-63 is on a loop near Cys-117. The distance between the two cysteines is compatible with formation of the Cys-117/Cys-63 intradisulfide bond. Analyses of MsrB sequences show that at least 37% of the MsrBs do not possess the recycling Cys-63. In the present study, it is shown that Cys-31 in the Xanthomonas campestris MsrB, which is located on another loop, can efficiently substitute for Cys-63. Such a result implies flexibility of the MsrB structures, at least of the loops on which Cys-31 or Cys-63 are located. The fact that about 25% of the putative MsrBs have no recycling cysteine supports other recycling processes in which thioredoxin is not operative.     

Identification and mapping of protein-protein interactions between gp32 and gp59 by cross-linking

The bacteriophage T4 59 protein (gp59) plays a vital role in recombination and replication by promoting the assembly of the gene 41 helicase (gp41) onto DNA, thus enabling replication as well as strand exchange in recombination. Loading of the helicase onto gp32 (the T4 single strand binding protein)-coated single-stranded DNA requires gp59 to remove gp32 and replace it with gp41. Cross-linking studies between gp32 and gp59 reveal an interaction between Cys-166 of gp32 and Cys-42 of gp59. Since Cys-166 lies in the DNA binding core domain of gp32, this interaction may affect the association of gp32 with DNA. In the presence of gp32 or DNA, gp59 is capable of forming a multimer consisting of at least five gp59 subunits. Kinetics studies suggest that gp59 and gp41 exist in a one-to-one ratio, predicting that gp59 is capable of forming a hexamer (Raney, K. D., Carver, T. E., and Benkovic, S. J. (1996) J. Biol. Chem. 271, 14074-14081). The C-terminal A-domain of gp32 is needed for gp59 oligomer formation. Cross-linking has established that gp59 can interact with gp32-A (a truncated form of gp32 lacking the A-domain) but cannot form higher species. The results support a model in which gp59 binds to gp32 on a replication fork, destabilizing the gp32-single-stranded DNA interaction concomitant with the oligomerization of gp59 that results in a switching of gp41 for gp32 at the replication fork.     

Amino acid sequence and disulfide-bridge location of canine haptoglobin.

The complete amino acid sequence and disulfide-bridge location of canine haptoglobin (Hp) were determined by analyzing various fragments produced chemically and/or enzymatically. Canine Hp consists of two light (L) and two heavy (H) chains with 83 and 245 amino acid residues, respectively. It has three potential oligosaccharide-binding sequences, Asn-X-Ser/Thr, one in the L chain and two in the H chain. All of them are glycosylated. Comparison of the amino acid sequences between canine Hp and human Hp shows 68 and 85% homology for L chains and H chains, respectively. About 20% of the canine L chain still retains a carboxyl-terminal arginine residue, which is completely removed during maturation in human L-chain. The half-cystine residue at position 15 in the L chain, which participates in the inter-L chain disulfide bridging in human Hp, has been replaced by a leucine residue in canine Hp. Therefore, an LH unit in canine Hp may be joined to another LH unit by a noncovalent (mainly hydrophobic) interaction to form the complete molecule. The disulfide bridges in canine Hp link Cys-34L to Cys-68L, Cys-72L to Cys-105H, Cys-148H to Cys-179H, and Cys-190H to Cys-220H, as in the case of human Hp.     

The functional role of cysteines in isopenicillin N synthase. Correlation of cysteine reactivities toward sulfhydryl reagents with kinetic properties of cysteine mutants

Isopenicillin N synthase (IPNS) from Cephalosporium acremonium contains 2 cysteine residues in positions 106 and 255 which are invariant in all IPNS sequences reported to date (Miller, J.R., and Ingolia, T.D. (1989) Mol. Microbiol. 3, 689-695). Although these residues have been postulated to play a role in catalysis (Samson, S.M., Chapman, J.L., Belagaje, R., Queener, S., and Ingolia, T.D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5705-5709) as well as enzyme inactivation (Perry, D., Abraham, E.P., and Baldwin, J.E. (1988) Biochem. J. 255, 345-351) little information exists regarding their oxidation state and reactivity. In this paper, the functions of these cysteines have been addressed by chemical modification techniques in combination with site-directed mutagenesis. In the intact wild type protein, both cysteines are inert toward 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetic acid. However, Cys-106, but not Cys-255, can be slowly modified by N-ethylmaleimide, and its modification is partially blocked by the presence of a substrate analog inhibitor. Complete modification of both cysteines by sulfhydryl reagents requires unfolding of the protein but not the presence of a disulfide reducing agent. The thiol content of IPNS is shown to be the same before and after exposing the enzyme to substrate even though during catalysis the enzyme is rapidly inactivated. The impact on catalysis due to alteration of the cysteines has been assessed using three site-specific mutants: Cys-106----Ser, Cys-255----Ser, and Cys-106,255----Ser. These mutant proteins have been purified as apoenzymes with the nature of the mutation verified by peptide mapping. The stoichiometry of metal required for activity remains as one equivalent of Fe2+/mol of enzyme in the mutants as in wild type IPNS. Compared with wild type, Cys-255----Ser shows a reduction in Vmax by 33%, and an increase in Km by 1.4-fold, while Cys-106----Ser and Cys-106,255----Ser, which have identical kinetic properties, exhibit a decrease in Vmax by 63% but an elevation of Km by 14-fold. The data presented demonstrate that 1) both cysteines are free thiols; 2) Cys-106 is more exposed than Cys-255; 3) substrate-induced inactivation is not caused by cysteine modification; 4) neither cysteine is absolutely essential for bond making or breaking events during catalysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antiparallel alignment of the two protomers of the regulatory subunit dimer of cAMP-dependent protein kinase I

The purified type I regulatory subunit of cAMP-dependent protein kinase is a dimeric protein, and the two protomers of the dimer are linked by two interchain disulfide bonds. The disulfide linkages that join these two polypeptide chains have been identified in order to provide a structural basis for the orientation of the two chains in the asymmetric dimer. Disulfide bonds were found to exist exclusively between Cys-16 and Cys-37, and this assignment, thus, establishes a general antiparallel alignment of the two chains. Two other homologous proteins, the type II regulatory subunit and the cGMP-dependent protein kinase also are dimeric proteins. In all three proteins, a relatively small, nonhomologous, amino-terminal segment of the polypeptide chain is essential for maintaining the dimeric aggregation state.     

Temperature-induced changes in the flexibility of the loop between SH1 (Cys-707) and SH3 (Cys-522) in myosin subfragment 1 detected by cross-linking

The ability of dibromobimane to cross-link SH1 (Cys-707) in the 21-kDa C-terminal segment to SH3 (Cys-522) in the 50-kDa middle segment of the myosin S1 heavy chain has been examined as a function of nucleotide binding and temperature. The results obtained indicate that, while the reagent rapidly reacts with SH1 at both 25 and 4 degrees C, its ability to cross-link to SH3 is highly dependent on temperature. At 25 degrees C, substantial cross-linking from monofunctionally labeled SH1 to SH3 occurs, in agreement with recent work of Mornet, Ue, and Morales (1985, Proc. Natl. Acad. Sci, USA 82, 1658-1662) and of Ue (1987, Biochemistry 26, 1889-1894) and with their conclusion that a loop, allowing SH1 and SH3 to reside at the cross-linking span of dibromobimane, preexists in the protein. At 4 degrees C, however, negligible amounts of cross-linking are observed whether or not a nucleotide is present, despite indications that SH1 is labeled rapidly by the reagent at this temperature. The inability to form this cross-link is not due to an alternate cross-link between monofunctionally labeled SH1 and another thiol in the 21-kDa segment. These results indicate that this loop exists at 25 degrees C and does not exist (or exists only transiently) at the lower temperature.