Permutation of the active site motif of tryparedoxin 2

Tryparedoxins (TXN) are thioredoxin-related proteins which, as trypanothione:peroxiredoxin oxidoreductases, constitute the trypanothione-dependent antioxidant defense and may also serve as substrates for ribonucleotide reductase in trypanosomatids. The active site motif of TXN2, 40WCPPCR45, of Crithidia fasciculata was mutated by site-directed mutagenesis and eight corresponding muteins were expressed in E. coli as terminally His-tagged proteins, purified to homogeneity by nickel chelate chromatography, and characterized in terms of specific activity, specificity and, if possible, kinetics. Exchange of Cys41 and Cys44 by serine yielded inactive products confirming their presumed involvement in catalysis. Exchange of Arg45 by aspartate resulted in loss of activity, suggesting an activation of active site cysteines by the positive charge of Arg45. Substitution of Trp40 by phenylalanine or tyrosine resulted in moderate decrease of specific activity, as did exchange of Pro42 by glycine. Kinetic analysis of these three muteins revealed that primarilythe reaction with trypanothione is affected by the mutations. Simulation of thioredoxin or glutaredoxin-like active sites in TXN2 (P42G and W40T/P43Y, respectively) did not result in thioredoxin or glutaredoxin-like activities. These data underscore that TXNs, although belonging to the thioredoxin superfamily, represent a group of enzymes distinct from thioredoxins and glutaredoxins in terms of specificity, and appear attractive as molecular targets for the design of trypanocidal compounds.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Oxidation of residue 45 mutant forms of pig deoxymyoglobin with [Fe(CN)6]3-.

The effect of replacement of the highly conserved Lys45 residue in pig myoglobin (Mb) with His, Ser, Glu, and Arg has been investigated. Rate constants/M-1 s-1 at 25 degrees C and pH 8.0, I = 0.100 M (NaCl), for the oxidation of deoxyMb with [Fe(CN)6]3- have been determined, and are for wild-type Lys45 (2.83 x 10(6)), His45 (1.02 x 10(6)), Ser45 (1.12 x 10(6)), Glu45 (0.87 x 10(6)), and Arg45 (3.06 x 10(6)). It is concluded that charge on the residue at position 45 has only a mild effect on reactivity, and that this is unlikely to be the site for electron transfer.     

Solution structure of marinostatin, a natural ester-linked protein protease inhibitor

Marinostatin is a unique protein protease inhibitor containing two ester linkages. We have purified a 12-residue marinostatin [MST(1-12), (1)FATMRYPSDSDE(12)] and determined the residues involved in the formation of the ester linkages and the solution structure by (1)H NMR spectroscopy and restrained molecular dynamics calculation. The two ester linkages of MST(1-12) are formed between hydroxyl and carboxyl groups, Thr(3)-Asp(9) and Ser(8)-Asp(11), indicating that MST(1-12) has two cyclic regions which are fused at the residues of Ser(8) and Asp(9). A strong NOE cross-peak between Tyr(6) H(alpha) and Pro(7) H(alpha) was observed, indicating that the Pro(7) residue takes a cis-conformation. Well-converged structures and hydrogen-deuterium experiments of MST(1-12) showed that the backbone NH proton of the P1'residue, Arg(5), is hydrogen-bonded to the carbonyl oxygen of the ester linkage between Thr(3) and Asp(9). To reveal the significance of the ester linkages, a marinostatin analogue, MST-2SS ((1)FACMRYPCCSCE(12)) with two disulfide bridges of Cys(3)-Cys(9) and Cys(8)-Cys(11), was also synthesized. The inhibitory activity of MST-2SS was as strong as that of MST(1-12), and the Pro(7) residue of MST-2SS also takes a cis-conformation. However, the exchange rate of the Arg(5) NH proton of MST-2SS was about 100 times faster than that of MST(1-12), and the structure calculation of MST-2SS was not converged on account of the small number of NOEs, indicating that MST-2SS takes a more flexible structure. The hydrogen acceptability of the ester linkage formed by the P2 position residue, Thr(3), is crucial for suppressing the fluctuation of the reactive site and sustaining the inhibitory activity, which enables marinostatin to be one of the smallest protease inhibitors in nature.     

Specificity and kinetics of a mitochondrial peroxiredoxin of Leishmania infantum

In Kinetoplastida, comprising the medically important parasites Trypanosoma brucei, T. cruzi, and Leishmania species, 2-Cys peroxiredoxins described to date have been shown to catalyze reduction of peroxides by the specific thiol trypanothione using tryparedoxin, a thioredoxin-related protein, as an immediate electron donor. Here we show that a mitochondrial peroxiredoxin from L. infantum (LimTXNPx) is also a tryparedoxin peroxidase. In an heterologous system constituted by nicotinamide adenine dinucleotide phosphate (NADPH), T. cruzi trypanothione reductase, trypanothione and Crithidia fasciculata tryparedoxin (CfTXN1 and CfTXN2), the recombinant enzyme purified from Escherichia coli as an N-terminally His-tagged protein preferentially reduces H(2)O(2) and tert-butyl hydroperoxide and less actively cumene hydroperoxide. Linoleic acid hydroperoxide and phosphatidyl choline hydroperoxide are poor substrates in the sense that they are reduced weakly and inhibit the enzyme in a concentration- and time-dependent way. Kinetic parameters deduced for LimTXNPx are a k(cat) of 37.0 s(-1) and K(m) values of 31.9 and 9.1 microM for CfTXN2 and tert-butyl hydroperoxide, respectively. Kinetic analysis indicates that LimTXNPx does not follow the classic ping-pong mechanism described for other TXNPx (Phi(1,2) = 0.8 s x microM(2)). Although the molecular mechanism underlying this finding is unknown, we propose that cooperativity between the redox centers of subunits may explain the unusual kinetic behavior observed. This hypothesis is corroborated by high-resolution electron microscopy and gel chromatography that reveal the native enzyme to preferentially exist as a homodecameric ring structure composed of five dimers.     

Crystal structure of a highly acidic neurotoxin from scorpion Buthus tamulus at 2.2A resolution reveals novel structural features

The crystal structure of a highly acidic neurotoxin from the scorpion Buthus tamulus has been determined at 2.2A resolution. The amino acid sequence determination shows that the polypeptide chain has 64 amino acid residues. The pI measurement gave a value of 4.3 which is one of the lowest pI values reported so far for a scorpion toxin. As observed in other alpha-toxins, it contains four disulphide bridges, Cys12-Cys63, Cys16-Cys36, Cys22-Cys46, and Cys26-Cys48. The crystal structure reveals the presence of two crystallographically independent molecules in the asymmetric unit. The conformations of two molecules are identical with an r.m.s. value of 0.3A for their C(alpha) tracings. The overall fold of the toxin is very similar to other scorpion alpha-toxins. It is a betaalphabetabeta protein. The beta-sheet involves residues Glu2-Ile6 (strand beta1), Asp32-Trp39 (strand beta3) and Val45-Val55 (strand beta4). The single alpha-helix formed is by residues Asn19-Asp28 (alpha2). The structure shows a trans peptide bond between residues 9 and 10 in the five-membered reverse turn Asp8-Cys12. This suggests that this toxin belongs to classical alpha-toxin subfamily. The surface features of the present toxin are highly characteristic, the first (A-site) has residues, Phe18, Trp38 and Trp39 that protrude outwardly presumably to interact with its receptor. There is another novel face (N-site) of this neurotoxin that contains several negatively charged residues such as, Glu2, Asp3, Asp32, Glu49 and Asp50 which are clustered in a small region of the toxin structure. On yet another face (P-site) in a triangular arrangement, with respect to the above two faces there are several positively charged residues, Arg58, Lys62 and Arg64 that also protrude outwardly for a potentially potent interaction with other molecules. This toxin with three strong features appears to be one of the most toxic molecules reported so far. In this sense, it may be a new subclass of neurotoxins with the largest number of hot spots.     

Selectivity of tryptophan residues in mediating photolysis of disulfide bridges in goat alpha-lactalbumin

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry. Peptides containing Cys120Cam, Cys61Cam, or Cys91Cam were detected, as well as two peptides containing a new Cys-Lys cross-link. In one, Cys6 was cross-linked to Lys122, while the cross-link in the second was either a Cys91-Lys79 or Cys73-Lys93 cross-link; however, the exact linkage could not be defined. The results demonstrate photolytic cleavage of the Cys6-Cys120, Cys61-Cys77, and Cys73-Cys91 disulfide bonds. While photolysis of Cys6-Cys120 and Cys73-Cys91 disulfide bonds in GLA has been reported, cleavage of the Cys61-Cys77 disulfide bonds has not been previously detected. To examine the contribution of the individual Trp residues, we constructed the GLA mutants, W26F, W60F, W104F, and W118F, by replacing single Trp residues with phenylalanine (Phe). The substitution of each Trp residue led to less thiol production compared to that for wild-type GLA, showing that each Trp residue in GLA contributed to the photolytic cleavage of disulfide bridges. The specificity was expressed by the nature of the reaction products. No cleavage of the Cys6-Cys120 disulfide bridge was detected when the W26F mutant was illuminated, and no cleavage of the Cys73-Cys91 disulfide bridge was seen following illumination of W26F or W104F. In contrast, Cys61Cam, resulting from the cleavage of the Cys61-Cys77 disulfide bridge, was found following illumination of any of the mutants.     

Interactions of human melanocortin 4 receptor with nonpeptide and peptide agonists

Specific interactions of human melanocortin-4 receptor (hMC4R) with its nonpeptide and peptide agonists were studied using alanine-scanning mutagenesis. The binding affinities and potencies of two synthetic, small-molecule agonists (THIQ, MB243) were strongly affected by substitutions in transmembrane alpha-helices (TM) 2, 3, 6, and 7 (residues Glu(100), Asp(122), Asp(126), Phe(261), His(264), Leu(265), and Leu(288)). In addition, a I129A mutation primarily affected the binding and potency of THIQ, while F262A, W258A, Y268A mutations impaired interactions with MB243. By contrast, binding affinity and potency of the linear peptide agonist NDP-MSH were substantially reduced only in D126A and H264A mutants. Three-dimensional models of receptor-ligand complexes with their agonists were generated by distance-geometry using the experimental, homology-based, and other structural constraints, including interhelical H-bonds and two disulfide bridges (Cys(40)-Cys(279), Cys(271)-Cys(277)) of hMC4R. In the models, all pharmacophore elements of small-molecule agonists are spatially overlapped with the corresponding key residues (His(6), d-Phe(7), Arg(8), and Trp(9)) of the linear peptide: their charged amine groups interact with acidic residues from TM2 and TM3, similar to His(6) and Arg(6) of NDP-MSH; their substituted piperidines mimic Trp(9) of the peptide and interact with TM5 and TM6, while the d-Phe aromatic rings of all three agonists contact with Leu(133), Trp(258), and Phe(261) residues.     

Relationship between temporary inhibition and structure of disulfide-linkage analogs of marinostatin, a natural ester-linked protein protease inhibitor.

A 12-residue marinostatin [MST(1-12): (1)FATMRYPSDSDE(12)] which contains two ester linkages of Thr(3)-Asp(9) and Ser(8)-Asp(11) strongly inhibits subtilisin. In order to study the relationship between the inhibitory activity, structure, and stability of MST, MST analogs were prepared by changing ester linkages to a disulfide linkages. The analogs without the disulfide linkage between 3 and 9 positions lost their inhibitory activity. The K(i) value of 1SS(C(3)-C(9)) ((1)FACMRYPSCSDE(12)), which has a single disulfide linkage of Cys(3)-Cys(9) was comparable with those of MST(1-12) and MST-2SS ((1)FACMRYPCCSCE(12)), a doubly linked analog of Cys(3)-Cys(9) and Cys(8)-Cys(11). However, 1SS(C(3)-C(9)) and MST-2SS showed temporary inhibition, but not MST(1-12): These analogs were inactivated after incubation with subtilisin for 30 min, and were specifically hydrolyzed at the reactive site. (1)H NMR study showed that 1SS(C(3)-C(9)) has two conformations, which contain a cis- (70%) or trans- (30%) Pro residue, while MST-2SS as well as MST(1-12) takes a single conformation containing only a cis-Pro residue. Hydrogen-deuterium exchange rate of the Arg(5) (P1') NH proton of the MST analogs was about 100 times faster than that of MST(1-12). These results indicate that the linkage between the positions 8 and 11 plays a role for fixing the cis-conformation of the Pro(7) residue, and that the linkage between 3 and 9 is indispensable for the inhibition, but not enough for stable protease-inhibitor complex.     

Amino acid sequences of two tryptic peptides from D(-)-beta-hydroxybutyrate dehydrogenase radiolabeled at essential carboxyl and sulfhydryl groups

D(-)beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria contains essential thiol and carboxyl groups. A tryptic BDH peptide labeled at an essential thiol with [3H]N-ethylmaleimide (NEM), and another tryptic peptide labeled at an essential carboxyl with N,N'-dicyclohexyl [14C]carbodiimide (DCCD), were isolated and sequenced. The peptide labeled with [3H]NEM had the sequence Met.Glu.Ser.Tyr.Cys*.Thr.Ser. Gly.Ser.Thr.Asp.Thr.Ser.Pro.Val.Ile.Lys. The label was at Cys. The same peptide was isolated from tryptic digests of BDH labeled at its nucleotide-binding site with the photoaffinity labeling reagent, arylazido- -[3-3H] alanyl-NAD. These results suggest that the essential thiol of BDH is located at its nucleotide-binding site, and agree with our previous observation that NAD and NADH protect BDH against inhibition by thiol modifiers. The [14C]DCCD-labeled peptide had the sequence Glu.Val.Ala.Glu*.Val. Asn. Leu.Trp.Gly.Thr.Val.Arg. DCCD appeared to modify the glutamic acid residue marked by an asterisk. Sequence analogies between these peptides and other proteins have been discussed.     

Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system. In vitro intragenic complementation: the roles of Arg126 in phosphoryl transfer and the C-terminal domain in dimerization

Enzyme I mutants of the Salmonella typhimurium phosphoenolpyruvate:sugar phosphotransferase system (PTS), which show in vitro intragenic complementation, have been identified as Arg126Cys (strain SB1690 ptsI34), Gly356Ser (strain SB1681 ptsI16), and Arg375Cys (strain SB1476 ptsI17). The mutation Arg126Cys is in the N-terminal HPr-binding domain, and complements Gly356Ser and Arg375Cys enzyme I mutations located in the C-terminal phosphoenolpyruvate(PEP)-binding domain. Complementation results in the formation of unstable heterodimers. None of the mutations alters the K(m) for HPr, which is phosphorylated by enzyme I. Arg126 is a conserved residue; the Arg126Cys mutation gives a V(max) of 0.04% wild-type, establishing a role in phosphoryl transfer. The Gly356Ser and Arg375Cys mutations reduce enzyme I V(max) to 4 and 2%, respectively, and for both, the PEP K(m) is increased from 0.1 to 3 mM. It is concluded that this activity was from the monomer, rather than the dimer normally found in assays of wild-type. In the presence of Arg126Cys enzyme, V(max) for Gly356Ser and Arg375Cys enzymes I increased 6- and 2-fold, respectively; the K(m) for PEP decreased to <10 microM, but the K(m) became dependent upon the stability of the heterodimer in the assay. Gly356 is conserved in enzyme I and pyruvate phosphate dikinase, which is a homologue of enzyme I, and this residue is part of a conserved sequence in the subunit interaction site. Gly356Ser mutation impairs enzyme I dimerization. The mutation Arg375Cys also impairs dimerization, but the equivalent residue in pyruvate phosphate dikinase is not associated with the subunit interaction site. A 37 000 Da, C-terminal domain of enzyme I has been expressed and purified; it dimerizes and complements Gly356Ser and Arg375Cys enzymes I proving that the association/dissociation properties of enzyme I are a function of the C-terminal domain.     

Isomers of epidermal growth factor with Ser --> Cys mutation at the N-terminal sequence: isomerization, stability, unfolding, refolding, and structure

The structure of human epidermal growth factor (EGF, 53 amino acids) comprises three distinct loops (A, B, and C) connected correspondingly by the three native disulfide bonds, Cys(6)-Cys(20), Cys(14)-Cys(31), and Cys(33)-Cys(42). The connection of Cys(6) and Cys(20) forming the N-terminal A loop is essential for the biological activity of EGF [Barnham et al. (1998) Protein Sci. 7, 1738-1749] and has also been shown to represent a major kinetic trap in the oxidative folding of EGF [Chang et al. (2001) J. Biol. Chem. 276, 4845-4852]. To further understand the chemical nature of this kinetic trap, we have prepared three EGF mutants each with a single Ser --> Cys mutation at Ser residues (Ser(2), Ser(4), and Ser(9)) flanking Cys(6). This allows competition between Cys(6) and mutated Cys(2), Cys(4), and Cys(9) to link with Cys(20) and to form EGF isomers containing different sizes of the A loop. The results show that, in the cases of EGF(S2C) and EGF(S4C), native Cys(6)-Cys(20) is favored over Cys(2)-Cys(20) and Cys(4)-Cys(20) by 4.5- and 9-fold, respectively, in the state of equilibrium. However, in the case of EGF(S9C), a non-native Cys(9)-Cys(20) is thermodynamically more stable than the native Cys(6)-Cys(20) by a free-energy difference (DeltaG degrees ) of 1.12 kcal/mol. Implications of these data in the formation of kinetic trap of EGF folding are discussed. Stabilized isomers of EGF were further generated from denaturation of wild-type and mutant EGF via the method of disulfide scrambling. Properties of these diverse isomers of EGF, including their isomerization, stability, unfolding, refolding, and disulfide structures, are described in this paper.     

Biosynthesis of pteridines. Reaction mechanism of GTP cyclohydrolase I

GTP cyclohydrolase I catalyses the hydrolytic release of formate from GTP followed by cyclization to dihydroneopterin triphosphate. The enzymes from bacteria and animals are homodecamers containing one zinc ion per subunit. Replacement of Cys110, Cys181, His112 or His113 of the enzyme from Escherichia coli by serine affords catalytically inactive mutant proteins with reduced capacity to bind zinc. These mutant proteins are unable to convert GTP or the committed reaction intermediate, 2-amino-5-formylamino-6-(beta-ribosylamino)-4(3H)-pyrimidinone 5'-triphosphate, to dihydroneopterin triphosphate. The crystal structures of GTP complexes of the His113Ser, His112Ser and Cys181Ser mutant proteins determined at resolutions of 2.5A, 2.8A and 3.2A, respectively, revealed the conformation of substrate GTP in the active site cavity. The carboxylic group of the highly conserved residue Glu152 anchors the substrate GTP, by hydrogen bonding to N-3 and to the position 2 amino group. Several basic amino acid residues interact with the triphosphate moiety of the substrate. The structure of the His112Ser mutant in complex with an undefined mixture of nucleotides determined at a resolution of 2.1A afforded additional details of the peptide folding. Comparison between the wild-type and mutant enzyme structures indicates that the catalytically active zinc ion is directly coordinated to Cys110, Cys181 and His113. Moreover, the zinc ion is complexed to a water molecule, which is in close hydrogen bond contact to His112. In close analogy to zinc proteases, the zinc-coordinated water molecule is suggested to attack C-8 of the substrate affording a zinc-bound 8R hydrate of GTP. Opening of the hydrated imidazole ring affords a formamide derivative, which remains coordinated to zinc. The subsequent hydrolysis of the formamide motif has an absolute requirement for zinc ion catalysis. The hydrolysis of the formamide bond shows close mechanistic similarity with peptide hydrolysis by zinc proteases.     

Evidence that Highly Conserved Residues of <Emphasis Type="Italic">Delonix regia</Emphasis> Trypsin Inhibitor Are Important for Activity

Delonix regia trypsin inhibitor (DrTI) consists of a single-polypeptide chain with a molecular mass of 22 kDa and containing two disulfide bonds (Cys44–Cys89 and Cys139–Cys149). Sequence comparison with other plant trypsin inhibitors of the Kunitz family reveals that DrTI contains a negatively charged residue (Glu68) at the reactive site rather than the conserved Arg or Lys found in other Kunitz-type trypsin inhibitors. Site-directed mutagenesis yielded five mutants containing substitutions at the reactive site and at one of the disulfide bonds. Assay of the recombinant proteins showed mutant Glu68Leu and Glu68Lys to have only 4–5% of the wild-type activity. These provide evidence that the Glu68 residue is the reactive site for DrTI and various other Kunitz-type trypsin inhibitors. The Cys139Gly mutant lost its inhibitory activity, whereas the Cys44Gly mutant did not, indicating that the second disulfide bond (Cys139–Cys149) is critical to DrTI inhibitory activity, while the first disulfide bond (Cys44–Cys89) is not required.     

Contributions of residue 45(CD3) and heme-6-propionate to the biomolecular and geminate recombination reactions of myoglobin

Overall association and dissociation rate constants were measured at 20 degrees C for O2, CO, and alkyl isocyanide binding to position 45 (CD3) mutants of pig and sperm whale myoglobins and to sperm whale myoglobin reconstituted with protoheme IX dimethyl ester. In pig myoglobin, Lys45(CD3) was replaced with Arg, His, Ser, and Glu; in sperm whale myoglobin, Arg45(CD3) was replaced with Ser and Gly. Intramolecular rebinding of NO, O2, and methyl isocyanide to Arg45, Ser45, Glu45, and Lys45(native) pig myoglobins was measured following 35-ps and 17-ns excitation pulses. The shorter, picosecond laser flash was used to examine ligand recombination from photochemically produced contact pairs, and the longer, nanosecond flash was used to measure the rebinding of ligands farther removed from the iron atom. Mutations at position 45 or esterification of the heme did not change significantly (less than or equal to 2-fold) the overall association rate constants for NO, CO, and O2 binding at room temperature. These data demonstrate unequivocally that Lys(Arg)45 makes little contribution to the outer kinetic barrier for the entry of diatomic gases into the distal pocket of myoglobin, a result that contradicts a variety of previous structural and theoretical interpretations. However, the rates of geminate recombination of NO and O2 and the affinity of myoglobin for O2 were dependent upon the basicity of residue 45. The series of substitutions Arg45, Lys45, Ser45, and Glu45 in pig myoglobin led to a 3-fold decrease in the initial rate for the intramolecular, picosecond rebinding of NO and 4-fold decrease in the geminate rate constant for the nanosecond rebinding of O2. (ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Interchain cysteine bridges control entry of progesterone to the central cavity of the uteroglobin dimer.

The progesterone-binding protein uteroglobin has been expressed in Escherichia coli in an unfused, soluble form. Like mature uteroglobin from rabbit endometrium (UG), the E.coli produced uteroglobin (UG1) dimerizes in vitro, forms an antiparallel dimer with Cys3-Cys69' and Cys69-Cys3' disulfide bonds and binds progesterone under reducing conditions. In order to analyze the dimerization and the reduction dependence of progesterone binding in more detail, we separately replaced cysteine 3 and cysteine 69 by serines. Under reducing conditions, both uteroglobin variants (UG1-3Ser and UG1-69Ser) bind progesterone with the same affinity as the wild-type suggesting that both cysteine residues are not directly involved in progesterone binding. In contrast to the wild-type protein, both cysteine variants also bind progesterone with high affinity in the absence of reducing agents. In addition, UG1-3Ser and UG1-69Ser both form covalently linked homodimers. Thus, unnatural Cys69-69' and Cys3-3' disulfide bonds exist in UG1-3Ser and UG1-69Ser, respectively. These data together with computer models based on X-ray diffraction data strongly support the idea that progesterone reaches its binding site located in an internal hydrophobic cavity via a hydrophobic tunnel along helices 1 and 4. Under non-reducing conditions the tunnel is closed by two disulfide bridges (Cys3-Cys69' and Cys69-Cys3') that lie in the most flexible region of the dimer. Reduction or replacement of a cysteine residue enables conformational changes that open the channel allowing progesterone to enter.     

Polymorphisms in base-excision repair and nucleotide-excision repair genes in relation to lung cancer risk

Polymorphisms in DNA repair genes may be associated with differences in DNA repair capacity, thereby influencing the individual susceptibility to smoking-related cancer. We investigated the association of 10 base-excision and nucleotide-excision repair gene polymorphisms (XRCC1 -77 T/C, Arg194Trp, Arg280His and Arg399Gln; APE1 Asp148Glu; OGG1 Ser326Cys; XPA -4 G/A; XPC PAT; XPD Asp312Asn and Lys751Gln) with lung cancer risk in Caucasians. Genotypes were determined by PCR-RFLP and PCR-single base extension assays in 110 lung cancer patients and 110 age- and sex-matched controls, and the results were analyzed using logistic regression adjusted for relevant covariates. A significant association between the APE1 Asp148Glu polymorphism and lung cancer risk was found, with adjusted odds ratios (OR) of 3.38 (p=0.001) for the Asp/Glu genotype and 2.39 (p=0.038) for the Glu/Glu genotype. Gene-smoking interaction analyses revealed a statistically significant interaction between cumulative cigarette smoking and the XRCC1 Arg399Gln and XPD Lys751Gln polymorphisms: these polymorphisms were significantly associated with lung cancer in nonsmokers and light smokers (<25 PY; OR=4.92, p=0.021 for XRCC1 399 Gln/Gln; OR=3.62, p=0.049 for XPD 751 Gln/Gln), but not in heavy smokers (> or =25 PY; OR=0.68, p=0.566 for XRCC1 399 Gln/Gln; OR=0.46, p=0.295 for XPD 751 Gln/Gln). Both the XRCC1 Arg194Trp and Arg280His as well as the OGG1 Ser326Cys heterozygous genotypes were associated with a significantly reduced risk for lung cancer (OR=0.32, p=0.024; OR=0.25, p=0.028; OR=0.51, p=0.033, respectively). No associations with lung cancer risk were found for the XRCC1 -77 T/C, the XPA -4 G/A and the XPC PAT polymorphisms. In conclusion, the APE1 Asp148Glu polymorphism is highly predictive for lung cancer, and cumulative cigarette smoking modifies the associations between the XRCC1 Arg399Gln and the XPD Lys751Gln polymorphisms and lung cancer risk.     

Assignment of the Disulfide Bridges in Bothropstoxin-I, a Myonecrotic Lys49 PLA2 Homolog from Bothrops jararacussu Snake Venom

Bothropstoxin-I (BthTX-I), a Lys49 phospholipase A₂ homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV₈) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV₈ digests: Cys50-Cys131, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cys105 by determination of the crystal structure, showing that BthTX-I disulfide bonds follow the normal pattern of group II PLA₂s.