Evidence that the methylesterase of bacterial chemotaxis may be a serine hydrolase.

CheB, the methylesterase of chemotactic bacteria, catalyzes the hydrolysis of glutamyl-methyl esters in bacterial chemoreceptor proteins. The two cysteines predicted by the amino acid sequence of CheB were replaced by alanine residues. The resulting mutants, Cys207-Ala, Cys309-Ala and a double cysteine mutant Cys207-Ala/Cys309-Ala, retained methylesterase activity, indicating that sulfhydryls are not crucial for CheB mediated catalysis. A homology search revealed a conserved serine active-site region between residues 162 and 166 which is homologous to the active-site region of acetylcholine esterases, suggesting that Ser164 of CheB is the active-site nucleophile. Oligonucleotide-directed mutagenesis was used to change the serine to a cysteine. This Ser164-Cys mutant had less than 2% of the wild-type activity. Unlike the serine proteinases which utilize a 'catalytic triad' mechanism, CheB does not have the conserved histidine and aspartic acid residues located in positions N-terminal to the active-site serine. In addition, CheB is not labeled with di-isopropylfluorophosphate, a potent inhibitor of other serine hydrolases. A novel mechanism is proposed for CheB involving substrate-assisted catalysis to account for these apparent anomalies.     

Characterization of recombinant human gastrointestinal glutathione peroxidase mutant produced in Escherichia coli

Abstract

Gastrointestinal glutathione peroxidase (GI-GPx, GPx2) is a selenium-dependent enzyme and regarded as the first line of defense against oxidative stress caused by ingested pro-oxidants or gut microbes. As the essential part of the catalytic site of GPx2, selenocysteine (Sec) is encoded by an in-frame UGA stop codon, which makes the expression of human GPx2 (hGPx2) using traditional recombinant DNA technology difficult. In order to produce bioactive recombinant hGPx2, the gene of hGPx2 was designed with the conversion of the codons for four cysteine (Cys) residues to the codons for serine (Ser) residues and the codon for Sec-40 was changed to the codon for Cys. This recombinant seleno-hGPx2 mutant was obtained using a single protein production system in a cysteine (Cys) auxotrophic strain, in which Sec was introduced into the protein via tRNA^Cys misleading. The activity of this mutant was in the same order of magnitude as that of hGPx4, but about one order of magnitude lower than that of hGPx1 and hGPx3. Further study showed that the mutant exhibited pH and temperature optima of 7.4 and 25°C, respectively. The results obtained from the kinetic analysis demonstrated that it followed a typical ping-pong mechanism similar to native GPx. As there was no report on the activity of purified GPx2, this research was valuable in recognizing native GPx2. In addition, a three-dimensional structure of seleno-hGPx2 mutant was constructed, which could facilitate further analysis of the role and the catalytic mechanism of native GPx2.     

Characterization of catalytic centre mutants of macrophage migration inhibitory factor (MIF) and comparison to Cys81Ser MIF.

Macrophage migration inhibitory factor (MIF) displays both cytokine and enzyme activities, but its molecular mode of action is still unclear. MIF contains three cysteine residues and we showed recently that the conserved Cys57-Ala-Leu-Cys60 (CALC) motif is critical for the oxidoreductase and macrophage-activating activities of MIF. Here we probed further the role of this catalytic centre by expression, purification, and characterization of the cysteine-->serine mutants Cys60Ser, Cys57Ser/Cys60Ser, and Cys81Ser of human MIF and of mutants Ala58Gly/Leu59Pro and Ala58Gly/Leu59His, containing a thioredoxin (Trx)-like and protein disulphide isomerase (PDI)-like dipeptide, respectively. The catalytic centre mutants formed inclusion bodies and the resultant mutant proteins Cys57Ser/Cys60Ser, Ala58Gly/Leu59Pro, and Als58Gly/Leu59His were only soluble in organic solvent or 6 m GdmHCl when reconstituted at concentrations above 1 microgram.mL-1. This made it necessary to devise new purification methods. By contrast, mutant Cys81Ser was soluble. Effects of pH, solvent, and ionic strength conditions on the conformation of the mutants were analysed by far-UV CD spectropolarimetry and mutant stability was examined by denaturant-induced unfolding. The mutants, except for mutant Cys81Ser, showed a close conformational similarity to wild-type (wt) MIF, and stabilization of the mutants was due mainly to acid pH conditions. Intramolecular disulphide bond formation at the CALC region was confirmed by near-UV CD of mutant Cys60Ser. Mutant Cys81Ser was not involved in disulphide bond formation, yet had decreased stability. Analysis in the oxidoreductase and a MIF-specific cytokine assay revealed that only substitution of the active site residues led to inactivation of MIF. Mutant Cys60Ser had no enzyme and markedly reduced cytokine activity, whereas mutant Cys81Ser was active in both tests. The Trx-like variant showed significant enzyme activity but was less active than wtMIF; PDI-like MIF was enzymatically inactive. However, both variants had full cytokine activity. Together with the low but nonzero cytokine activity of mutant Cys60Ser, this indicated that the cytokine activity of MIF may not be tightly regulated by redox effects or that a distinguishable receptor mechanism exists. This study provides evidence for a role of the CALC motif in the oxidoreductase and cytokine activities of MIF, and suggests that Cys81 could mediate conformational effects. Availability and characterization of the mutants should greatly aid in the further elucidation of the mechanism of action of the unusual cytokine MIF.     

Thermostabilization of recombinant human and bovine CuZn superoxide dismutases by replacement of free cysteines

Human CuZn superoxide dismutase (HSOD) has two free cysteines: a buried cysteine (Cys6) located in a beta-strand, and a solvent accessible cysteine (Cys111) located in a loop region. The highly homologous bovine enzyme (BSOD) has a single buried Cys6 residue. Cys6 residues in HSOD and BSOD were replaced by alanine and Cys111 residues in HSOD by serine. The mutant enzymes were expressed and purified from yeast and had normal specific activities. The relative resistance of the purified proteins to irreversible inactivation of enzymatic activity by heating at 70 degrees C was HSOD Ala6 Ser111 greater than BSOD Ala6 Ser109 greater than BSOD Cys6 Ser109 (wild type) greater than HSOD Ala6 Cys111 greater than HSOD Cys6 Ser111 greater than HSOD Cys111 (wild type). In all cases, removal of a free cysteine residue increased thermostability.     

Properties of Cys21-mutated muscle acylphosphatases

Cys21 is an invariant residue in muscle acylphosphatases, but is absent in the erythrocyte isozymes. To assess the importance of this residue in the muscle isozymes for catalytic, structural, and stability properties, two gene mutants have been prepared by oligonucleotide-directed mutagenesis and expressed inEscherichia coli cells; in these mutants, the codon for Cys21 was replaced by those for Ser and Ala, respectively. The two mutant enzymes, purified by immunoaffinity chromatography, showed kinetic and structural properties similar to those of the wild-type recombinant enzyme; however, the specific activity of the two mutants, especially that of the C21A mutant, was lower. The urea and thermal stabilities of the mutant enzymes were reduced with respect to those of the wild-type form, contrary to the susceptibility to inactivation by mercuric ions. The reported data support the possibility that Cys21 is involved in the stabilization of the enzyme active-site conformation.     

Sorsby's fundus dystrophy tissue inhibitor of metalloproteinases-3 (TIMP-3) mutants have unimpaired matrix metalloproteinase inhibitory activities, but affect cell adhesion to the extracellular matrix.

The TIMP family of matrix metalloproteinase inhibitors consists of four members, of which TIMP-1, -2 and -4 are secreted, freely diffusible proteins, whereas TIMP-3 is ECM-associated. Mutations in the TIMP3 gene have been linked to Sorsby's fundus dystrophy (SFD), an autosomal dominant inherited retinal degenerative disease that leads to blindness. The SFD mutations characterized result in introduction of an unpaired cysteine residue in the C-terminal domain of TIMP-3. We have expressed four SFD mutant TIMP-3 proteins in baby hamster kidney (BHK) cells and evaluated their characteristics alongside wild-type TIMP-3. Analysis of the mutant proteins (Ser156Cys, Gly167Cys, Tyr168Cys and Ser181Cys) by SDS-PAGE and reverse zymography revealed that each of the mutants retained gelatinase A and gelatinase B inhibitory activity, and were localized to the ECM. Association rate constants for Ser156Cys TIMP-3 with gelatinase-A, gelatinase-B, stromelysin-1 and collagenase-3 were only moderately reduced compared to wild-type TIMP-3. However, all of the mutants displayed aberrant protein-protein interactions, resulting in the presence of additional proteins or complexes in ECM preparations. Two of the mutants (Ser156Cys and Ser181Cys) showed a marked propensity to form multiple higher molecular-weight complexes that retained TIMP activity on reverse zymography. Expression of the SFD mutant TIMP-3 (and to a lesser extent, wild-type TIMP-3) proteins in BHK cells conferred increased cell adhesiveness to the ECM. Our findings indicate that the pathogenesis of Sorsby's fundus dystrophy cannot be attributed to a failure to localize SFD TIMP-3 proteins to the ECM or defects in MMP inhibition, but may involve the formation of aberrant TIMP-3-containing protein complexes and altered cell adhesion.     

The isocitrate dehydrogenase kinase/phosphatase from Escherichia coli is highly sensitive to in-vitro oxidative conditions role of cysteine67 and cysteine108 in the formation of a disulfide-bonded homodimer.

Isocitrate dehydrogenase kinase/phosphatase (IDHK/P) is a homodimeric enzyme which controls the oxidative metabolism of Escherichia coli, and exibits a high intrinsic ATPase activity. When subjected to electrophoresis under nonreducing conditions, the purified enzyme migrates partially as a dimer. The proportion of the dimer over the monomer is greatly increased by treatment with cupric 1,10 phenanthrolinate or 5,5'-dithio-bis(2-nitrobenzoic acid), and fully reversed by dithiothreitol, indicating that covalent dimerization is produced by a disulfide bond. To identify the residue(s) involved in this intermolecular disulfide-bond, each of the eight cysteines of the enzyme was individually mutated into a serine. It was found that, under nonreducing conditions, the electrophoretic patterns of all corresponding mutants are identical to that of the wild-type, except for the Cys67-->Ser which migrates exclusively as a monomer and for the Cys108-->Ser which migrates preferentially as a dimer. Furthermore, in contrast to the wild-type enzyme and all the other mutants, the Cys67-->Ser mutant still migrates as a monomer after treatment with cupric 1,10 phenanthrolinate. This result indicates that the intermolecular disulfide bond involves only Cys67 in each IDHK/P wild-type monomer. This was further supported by mass spectrum analysis of the tryptic peptides derived from either the cupric 1,10 phenanthrolinate-treated wild-type enzyme or the native Cys108-->Ser mutant, which show that they both contain a Cys67-Cys67 disulfide bond. Moreover, both the cupric 1,10 phenanthrolinate-treated wild-type enzyme and the native Cys108-->Ser mutant contain another disulfide bond between Cys356 and Cys480. Previous results have shown that this additional Cys356-Cys480 disulfide bond is intramolecular [Oudot, C., Jault, J.-M., Jaquinod, M., Negre, D., Prost, J.-F., Cozzone, A.J. & Cortay, J.-C. (1998) Eur. J. Biochem. 258, 579-585].     

Mechanistic studies on beta-ketoacyl thiolase from Zoogloea ramigera: identification of the active-site nucleophile as Cys89, its mutation to Ser89, and kinetic and thermodynamic characterization of wild-type and mutant enzymes

Thiolase proceeds via covalent catalysis involving an acetyl-S-enzyme. The active-site thiol nucleophile is identified as Cys89 by acetylation with [14C]acetyl-CoA, rapid denaturation, tryptic digestion, and sequencing of the labeled peptide. The native acetyl enzyme is labile to hydrolytic decomposition with t 1/2 of 2 min at pH 7, 25 degrees C. Cys89 has been converted to the alternate nucleophile Ser89 by mutagenesis and the C89S enzyme overproduced, purified, and assessed for activity. The Ser89 enzyme retains 1% of the Vmax of the Cys89 enzyme in the direction of acetoacetyl-CoA thiolytic cleavage and 0.05% of the Vmax in the condensation of two acetyl-CoA molecules. A covalent acetyl-O-enzyme intermediate is detected on incubation with [14C]acetyl-CoA and isolation of the labeled Ser89-containing tryptic peptide. Comparisons of the Cys89 and Ser89 enzymes have been made for kinetic and thermodynamic stability of the acetyl enzyme intermediates both by isolation and by analysis of [32P]CoASH/acetyl-CoA partial reactions and for rate-limiting steps in catalysis with trideuterioacetyl-CoA.     

Engineered fatty acid biosynthesis in Streptomyces by altered catalytic function of beta-ketoacyl-acyl carrier protein synthase III

The Streptomyces glaucescens beta-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) initiates straight- and branched-chain fatty acid biosynthesis by catalyzing the decarboxylative condensation of malonyl-ACP with different acyl-coenzyme A (CoA) primers. This KASIII has one cysteine residue, which is critical for forming an acyl-enzyme intermediate in the first step of the process. Three mutants (Cys122Ala, Cys122Ser, Cys122Gln) were created by site-directed mutagenesis. Plasmid-based expression of these mutants in S. glaucescens resulted in strains which generated 75 (Cys122Ala) to 500% (Cys122Gln) more straight-chain fatty acids (SCFA) than the corresponding wild-type strain. In contrast, plasmid-based expression of wild-type KASIII had no effect on fatty acid profiles. These observations are attributed to an uncoupling of the condensation and decarboxylation activities in these mutants (malonyl-ACP is thus converted to acetyl-ACP, a SCFA precursor). Incorporation experiments with perdeuterated acetic acid demonstrated that 9% of the palmitate pool of the wild-type strain was generated from an intact D(3) acetyl-CoA starter unit, compared to 3% in a strain expressing the Cys122Gln KASIII. These observations support the intermediacy of malonyl-ACP in generating the SCFA precursor in a strain expressing this mutant. To study malonyl-ACP decarboxylase activity in vitro, the KASIII mutants were expressed and purified as His-tagged proteins in Escherichia coli and assayed. In the absence of the acyl-CoA substrate the Cys122Gln mutant and wild-type KASIII were shown to have comparable decarboxylase activities in vitro. The Cys122Ala mutant exhibited higher activity. This activity was inhibited for all enzymes by the presence of high concentrations of isobutyryl-CoA (>100 microM), a branched-chain fatty acid biosynthetic precursor. Under these conditions the mutant enzymes had no activity, while the wild-type enzyme functioned as a ketoacyl synthase. These observations indicate the likely upper and lower limits of isobutyryl-CoA and related acyl-CoA concentrations within S. glaucescens.     

Identification of the zinc ligands in cobalamin-independent methionine synthase (MetE) from Escherichia coli

Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. It contains 1 equiv of zinc that is essential for its catalytic activity. Extended X-ray absorption fine structure analysis of the zinc-binding site has suggested tetrahedral coordination with two sulfur (cysteine) and one nitrogen or oxygen ligands provided by the enzyme and an exchangeable oxygen or nitrogen ligand that is replaced by the homocysteine thiol group in the enzyme-substrate complex [González, J. C., Peariso, K., Penner-Hahn, J. E., and Matthews, R. G. (1996) Biochemistry 35, 12228-34]. Sequence alignment of MetE homologues shows that His641, Cys643, and Cys726 are the only conserved residues. We report here the construction, expression, and purification of the His641Gln, Cys643Ser, and Cys726Ser mutants of MetE. Each mutant displays significantly impaired activity and contains less than 1 equiv of zinc upon purification. Furthermore, each mutant binds zinc with lower binding affinity (K(a) approximately 10(14) M(-)(1)) compared to the wild-type enzyme (K(a) > 10(16) M(-)(1)). All the MetE mutants are able to bind homocysteine. X-ray absorption spectroscopy analysis of the zinc-binding sites in the mutants indicates that the four-coordinate zinc site is preserved but that the ligand sets are changed. Our results demonstrate that Cys643 and Cys726 are two of the zinc ligands in MetE from E. coli and suggest that His641 is a third endogenous ligand. The effects of the mutations on the specific activities of the mutant proteins suggest that zinc and homocysteine binding alone are not sufficient for activity; the chemical nature of the ligands is also a determining factor for catalytic activity in agreement with model studies of the alkylation of zinc-thiolate complexes.     

Homodimeric mitochondrial phosphate transport protein. Transient subunit/subunit contact site between the transport relevant transmembrane helices A

The three Cys of the yeast (Saccharomyces cerevisiae) mitochondrial phosphate transport protein (PTP) subunit were replaced with Ser. The seven mutants (single, double, and complete Cys replacements) were expressed in yeast, and the homodimeric mutant PTPs were purified from the mitochondria and reconstituted. The pH gradient-dependent net phosphate (Pi) transport uptake rates (initial conditions: 1 mM [Pi]e, pHe 6.80; 0 mM [Pi]i, pHi 8.07) catalyzed by these reconstituted mutants are similar to those of the wild-type protein and range from 15 to 80 micromol Pi/min mg PTP protein. Aerobic media inhibit only the Pi uptake rates catalyzed by PTPs with the conserved (yeast and bovine) Cys28. This inhibition in the proteoliposomes is 84-95% and can be completely reversed by dithiothreitol. Transport by the wild type as well as by all mutant proteins with Cys28 is more than 90% inhibited by mersalyl. Transport catalyzed by mutant proteins with only Cys300 or only Cys134 is less sensitive, and that catalyzed by the no Cys mutant shows 40% inhibition by mersalyl. When dithiothreitol is removed from purified single Cys mutant proteins, only the mutant protein with Cys28 appears as a homodimer in a nonreducing SDS polyacrylamide gel. Thus, the function relevant transmembrane helix A, with Cys 28 about equidistant from the two inner membrane surfaces, is in close contact with parts of transmembrane helix A of the other subunit in the functional homodimeric PTP. The results identify for the first time not only a transmembrane helix contact site between the two subunits of a homodimeric mitochondrial transport protein but also a contact site that if locked into position blocks transport. The results are related to two available secondary transporter structures (lactose permease, glycerol-3-phosphate transporter) as well as to a low resolution projection structure and a high resolution structure of monomers of inhibitor ADP/ATP carrier complexes.     

The iron-sulfur center of biotin synthase: site-directed mutants

Biotin synthase contains an essential [4Fe-4S]+ cluster that is thought to provide an electron for the cleavage of S-adenosylmethionine, a cofactor required for biotin formation. The conserved cysteine residues Cys53, Cys57 and Cys60 have been proposed as ligands to the [4Fe-4S] cluster. These residues belong to a C-X3-C-X2-C motif which is also found in pyruvate formate lyase-activating enzyme, lysine 2,3-aminomutase and the anaerobic ribonucleotide reductase-activating component. To investigate the role of the cysteine residues, Cys-->Ala mutants of the eight cysteine residues of Escherichia coli biotin synthase were prepared and assayed for activity. Our results show that six cysteines are important for biotin formation. Only two mutant proteins, C276A and C288A, closely resembled the wild-type protein, indicating that the corresponding cysteines are not involved in iron chelation and biotin formation. The six other mutant proteins, C53A, C57A, C60A, C97A, C128A and C188A, were inactive but capable of assembling a [4Fe-4S] cluster, as shown by M?ssbauer spectroscopy. The C53A, C57A and C60A mutant proteins are unique in that their cluster could not undergo reduction to the [4Fe-4S]+ state, as shown by EPR and M?ssbauer spectroscopy. On this basis and by analogy with pyruvate formate lyase-activating enzyme and the anaerobic ribonucleotide reductase-activating component, it is suggested that the corresponding cysteines coordinate the cluster even though one cannot fully exclude the possibility that other cysteines play that role as well. Therefore it appears that for activity biotin synthase absolutely requires cysteines that are not involved in iron chelation.     

Different Catalytic Mechanisms in Mammalian Selenocysteine- and Cysteine-Containing Methionine-R-Sulfoxide Reductases

Selenocysteine (Sec) is found in active sites of several oxidoreductases in which this residue is essential for catalytic activity. However, many selenoproteins have fully functional orthologs, wherein cysteine (Cys) occupies the position of Sec. The reason why some enzymes evolve into selenoproteins if the Cys versions may be sufficient is not understood. Among three mammalian methionine-R-sulfoxide reductases (MsrBs), MsrB1 is a Sec-containing protein, whereas MsrB2 and MsrB3 contain Cys in the active site, making these enzymes an excellent system for addressing the question of why Sec is used in biological systems. In this study, we found that residues, which are uniquely conserved in Cys-containing MsrBs and which are critical for enzyme activity in MsrB2 and MsrB3, were not required for MsrB1, but increased the activity of its Cys mutant. Conversely, selenoprotein MsrB1 had a unique resolving Cys reversibly engaged in the selenenylsulfide bond. However, this Cys was not necessary for activities of either MsrB2, MsrB3, or the Cys mutant of MsrB1. We prepared Sec-containing forms of MsrB2 and MsrB3 and found that they were more than 100-fold more active than the natural Cys forms. However, these selenoproteins could not be reduced by the physiological electron donor, thioredoxin. Yet, insertion of the resolving Cys, which was conserved in MsrB1, into the selenoprotein form of MsrB3 restored the thioredoxin-dependent activity of this enzyme. These data revealed differences in catalytic mechanisms between selenoprotein MsrB1 and non-selenoproteins MsrB2 and MsrB3, and identified catalytic advantages and disadvantages of Sec- and Cys-containing proteins. The data also suggested that Sec- and Cys-containing oxidoreductases require distinct sets of active-site features that maximize their catalytic efficiencies and provide strategies for protein design with improved catalytic properties.     

Different catalytic mechanisms in mammalian selenocysteine- and cysteine-containing methionine-R-sulfoxide reductases

Selenocysteine (Sec) is found in active sites of several oxidoreductases in which this residue is essential for catalytic activity. However, many selenoproteins have fully functional orthologs, wherein cysteine (Cys) occupies the position of Sec. The reason why some enzymes evolve into selenoproteins if the Cys versions may be sufficient is not understood. Among three mammalian methionine-R-sulfoxide reductases (MsrBs), MsrB1 is a Sec-containing protein, whereas MsrB2 and MsrB3 contain Cys in the active site, making these enzymes an excellent system for addressing the question of why Sec is used in biological systems. In this study, we found that residues, which are uniquely conserved in Cys-containing MsrBs and which are critical for enzyme activity in MsrB2 and MsrB3, were not required for MsrB1, but increased the activity of its Cys mutant. Conversely, selenoprotein MsrB1 had a unique resolving Cys reversibly engaged in the selenenylsulfide bond. However, this Cys was not necessary for activities of either MsrB2, MsrB3, or the Cys mutant of MsrB1. We prepared Sec-containing forms of MsrB2 and MsrB3 and found that they were more than 100-fold more active than the natural Cys forms. However, these selenoproteins could not be reduced by the physiological electron donor, thioredoxin. Yet, insertion of the resolving Cys, which was conserved in MsrB1, into the selenoprotein form of MsrB3 restored the thioredoxin-dependent activity of this enzyme. These data revealed differences in catalytic mechanisms between selenoprotein MsrB1 and non-selenoproteins MsrB2 and MsrB3, and identified catalytic advantages and disadvantages of Sec- and Cys-containing proteins. The data also suggested that Sec- and Cys-containing oxidoreductases require distinct sets of active-site features that maximize their catalytic efficiencies and provide strategies for protein design with improved catalytic properties.     

Stability of wild-type and mutant RTEM-1 beta-lactamases: effect of the disulfide bond

Uniquely among class A beta-lactamases, the RTEM-1 and RTEM-2 enzymes contain a single disulfide bond between Cys 77 and Cys 123. To study the possible role of this naturally occurring disulfide in stabilizing RTEM-1 beta-lactamase and its mutants at residue 71, this bond was removed by introducing a Cys 77----Ser mutation. Both the wild-type enzyme and the single mutant Cys 77----Ser confer the same high levels of resistance to ampicillin in vivo to Escherichia coli; at 30 degrees C the specific activity of purified Cys 77----Ser mutant is also the same as that of the wild-type enzyme. Also, neither wild-type enzyme nor the Cys 77----Ser mutant is inactivated by brief exposure to p-hydroxymercuribenzoate. However, above 40 degrees C the mutant enzyme is less stable than wild-type enzyme. After introduction of the Cys 77----Ser mutation, none of the double mutants (containing the second mutations at residue 71) confer resistance to ampicillin in vivo at 37 degrees C; proteins with Ala, Val, Leu, Ile, Met, Pro, His, Cys, and Ser at residue 71 confer low levels of resistance to ampicillin in vivo at 30 degrees C. The use of electrophoretic blots stained with antibodies against beta-lactamase to analyze the relative quantities of mutant proteins in whole-cell extracts of E. coli suggests that all 19 of the doubly mutant enzymes are proteolyzed much more readily than their singly mutant analogues (at Thr 71) that contain a disulfide bond.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nickel superoxide dismutase: structural and functional roles of Cys2 and Cys6

Nickel superoxide dismutase (NiSOD) is unique among the family of superoxide dismutase enzymes in that it coordinates Cys residues (Cys2 and Cys6) to the redox-active metal center and exhibits a hexameric quaternary structure. To assess the role of the Cys residues with respect to the activity of NiSOD, mutations of Cys2 and Cys6 to Ser (C2S-NiSOD, C6S-NiSOD, and C2S/C6S-NiSOD) were carried out. The resulting mutants do not catalyze the disproportionation of superoxide, but retain the hexameric structure found for wild-type NiSOD and bind Ni(II) ions in a 1:1 stoichiometry. X-ray absorption spectroscopic studies of the Cys mutants revealed that the nickel active-site structure for each mutant resembles that of C2S/C6S-NiSOD and demonstrate that mutation of either Cys2 or Cys6 inhibits coordination of the remaining Cys residue. Mutation of one or both Cys residue(s) in NiSOD induces the conversion of the low-spin Ni(II) site in the native enzyme to a high-spin Ni(II) center in the mutants. This result indicates that coordination of both Cys residues is required to generate the native low-spin configurations and maintain catalytic activity. Analysis of the quaternary structure of the Cys mutants by differential scanning calorimetry, mass spectrometry, and size-exclusion chromatography revealed that the Cys ligands, particularly Cys2, are also important for stabilizing the hexameric quaternary structure of the native enzyme.     

Activation of factor V during intrinsic and extrinsic coagulation. Inhibition by heparin, hirudin and D-Phe-Pro-Arg-Ch2Cl.

Ser130, Asp131 and Asn132 ('SDN') are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.     

Site-directed mutagenesis studies on the recombinant thioesterase domain of chicken fatty acid synthase expressed in Escherichia coli

The animal fatty acid synthase is a multifunctional protein with a subunit molecular weight of 260,000. We recently reported the expression and characterization of the acyl carrier protein and thioesterase domains of the chicken liver fatty acid synthase in Escherichia coli. In order to gain insight into the mechanism of action of the thioesterase domain, we have replaced the putative active site serine 101 with alanine and cysteine and the conserved histidine 274 with alanine by site-directed mutagenesis. While both the Ser101----Ala and His274----Ala mutant proteins were inactive, the Ser101----Cys mutant enzyme (thiol-thioesterase) retained considerable activity, but the properties of the enzyme were changed from an active serine esterase to an active cysteine esterase, providing strong evidence for the role of Ser101 as the active site nucleophile. In order to further probe into the role of His274, a double mutant was constructed containing both the Ser101----Cys and the His274----Ala mutations. The double-mutant protein was inactive and exhibited diminished reactivity of the Cys-SH to iodoacetamide as compared to that of the Ser101----Cys-thioesterase, suggesting a role of His274 as a general base in withdrawing the proton from the Cys-SH in the thiol-thioesterase or Ser101 in the wild-type enzyme. Incubation of the recombinant thioesterases with [1-14C] palmitoyl-CoA resulted in the incorporation of [1-14C] palmitoyl into the enzyme only in the double mutant, suggesting that Cys-SH of the double mutant is reactive enough to form the palmitoyl-S-enzyme intermediate. This intermediate is not hydrolyzed because of the lack of His274, which is required for the attack of H2O on the acyl enzyme. These results suggest that the catalytic mechanism of the thioesterases may be similar to that of the serine proteases and lipases, which employ a serine-histidine-aspartic acid catalytic triad as part of their catalytic mechanism.     

Reduced activity of bamhi variants c54i, c64w, and c54d/c64r is consistent with the substrate-assisted catalysis model

Three specific mutants, C54I, C54W, and a double-mutant C54D:C64R of restriction endonuclease BamHI, were generated and studied to investigate the role, if any, of the 54th and 64th cysteine residues in the catalysis of BamHI. The mutation was achieved using the megaprimer approach for PCR. The mutant genes were cloned and characterized by sequencing. The mutant and the wild-type proteins were expressed and purified and their kinetic parameters were determined using short synthetic oligonucleotides as substrates. All mutants had higher K(m) values than that of the wild-type enzyme suggesting a decrease in the affinity of the enzyme for its substrate. The mutant protein C54W showed significant changes in the CD spectra vis-a-vis wild-type enzyme and had the lowest K(m)/K(cat) value among the mutants indicative of changes in the secondary structure of the protein. The melting curves of the mutant proteins overlapped that of the wild-type enzyme. Analysis of the K(cat) values in the context of cocrystal structure suggests that the effect of Cys54 mutation is probably through the perturbation of the local structure whereas reduced activity of the double mutant is consistent with the substrate-assisted catalysis mechanism.     

Serine incorporation into the selenocysteine moiety of glutathione peroxidase

The selenium in mammalian glutathione peroxidase is present as a selenocysteine ([Se]Cys) moiety incorporated into the peptide backbone 41-47 residues from the N-terminal end. To study the origin of the skeleton of the [Se]Cys moiety, we perfused isolated rat liver with 14C- or 3H-labeled amino acids for 4 h, purified the GSH peroxidase, derivatized the [Se]Cys in GSH peroxidase to carboxymethylselenocysteine ([Se]Cys(Cm)), and determined the amino acid specific activity. Perfusion with [14C]cystine resulted in [14C]cystine incorporation into GSH peroxidase without labeling [Se]Cys(Cm), indicating that cysteine is not a direct precursor for [Se]Cys. [14C]Serine perfusion labeled serine, glycine (the serine hydroxymethyltransferase product), and [Se]Cys(Cm) in purified GSH peroxidase, whereas [3-3H]serine perfusion only labeled serine and [Se]Cys(Cm), thus demonstrating that the [Se]Cys in GSH peroxidase is derived from serine. The similar specific activities of serine and [Se]Cys(Cm) strongly suggest that the precursor pool of serine used for [Se] Cys synthesis is the same or similar to the serine pool used for acylation of seryl-tRNAs.