Physical association of starch biosynthetic enzymes with starch granules of maize endosperm. Granule-associated forms of starch synthase I and starch branching enzyme II

Antibodies were used to probe the degree of association of starch biosynthetic enzymes with starch granules isolated from maize (Zea mays) endosperm. Graded washings of the starch granule, followed by release of polypeptides by gelatinization in 2% sodium dodecyl sulfate, enables distinction between strongly and loosely adherent proteins. Mild aqueous washing of granules resulted in near-complete solubilization of ADP-glucose pyrophosphorylase, indicating that little, if any, ADP-glucose pyrophosphorylase is granule associated. In contrast, all of the waxy protein plus significant levels of starch synthase I and starch branching enzyme II (BEII) remained granule associated. Stringent washings using protease and detergent demonstrated that the waxy protein, more than 85% total endosperm starch synthase I protein, and more than 45% of BEII protein were strongly associated with starch granules. Rates of polypeptide accumulation within starch granules remained constant during endosperm development. Soluble and granule-derived forms of BEII yielded identical peptide maps and overlapping tryptic fragments closely aligned with deduced amino acid sequences from BEII cDNA clones. These observations provide direct evidence that BEII exits as both soluble and granule-associated entities. We conclude that each of the known starch biosynthetic enzymes in maize endosperm exhibits a differential propensity to associate with, or to become irreversibly entrapped within, the starch granule.     

Prevalence and determinants of Helicobacter pylori infection in preschool children: a population-based study from Germany

Branching enzyme (BE) belongs to the amylolytic family which contains four highly conserved regions. These regions are proposed to play an important role in catalysis as they are thought to be necessary for catalysis and/or binding the substrate. Only one arginine residue was found to be conserved in a catalytic center at the same position in all known sequences of BEs from various species as well as in the alpha-amylase enzyme family. In mBEII, a conserved Arg residue 384 is in catalytic region 2. We have used site-directed mutagenesis of the Arg-384 residue in order to study its possible role in BE. Previous chemical modification studies (H. Cao and J. Preiss, 1996, J. Prot. Chem. 15, 291-304) suggest that it may play a role in substrate binding. Replacement of Arg-384 by Ala, Ser, Gln, and Glu in the active site caused almost total inactivation. However, a conservative mutation of the conserved Arg-384 by Lys resulted in some residual activity, approximately 5% of the wild-type enzyme. The kinetics of the purified mutant R384K enzyme were investigated and no large effect on the Km of the substrate amylose for BE was observed. Thus, these results suggest that conserved Arg residue 384 in mBEII plays an important role in the catalytic function of BEs but may not be directly involved in substrate binding.     

Vitamin K2 (menaquinone) biosynthesis in Escherichia coli: evidence for the presence of an essential histidine residue in o-succinylbenzoyl coenzyme A synthetase

o-Succinylbenzoyl coenzyme A (OSB-CoA) synthetase, when treated with diethylpyrocarbonate (DEP), showed a time-dependent loss of enzyme activity. The inactivation follows pseudo-first-order kinetics with a second-order rate constant of 9.2 x 10(-4) +/- 1.4 x 10(-4) microM(-1) min(-1). The difference spectrum of the modified enzyme versus the native enzyme showed an increase in A242 that is characteristic of N-carbethoxyhistidine and was reversed by treatment with hydroxylamine. Inactivation due to nonspecific secondary structural changes in the protein and modification of tyrosine, lysine, or cysteine residues was ruled out. Kinetics of enzyme inactivation and the stoichiometry of histidine modification indicate that of the eight histidine residues modified per subunit of the enzyme, a single residue is responsible for the enzyme activity. A plot of the log reciprocal of the half-time of inactivation against the log DEP concentration further suggests that one histidine residue is involved in the catalysis. Further, the enzyme was partially protected from inactivation by either o-succinylbenzoic acid (OSB), ATP, or ATP plus Mg2+ while inactivation was completely prevented by the presence of the combination of OSB, ATP, and Mg2+. Thus, it appears that a histidine residue located at or near the active site of the enzyme is essential for activity. When His341 present in the previously identified ATP binding motif was mutated to Ala, the enzyme lost 65% of its activity and the Km for ATP increased 5.4-fold. Thus, His341 of OSB-CoA synthetase plays an important role in catalysis since it is probably involved in the binding of ATP to the enzyme.     

The treatment of flexion contracture of the knee in myelomeningocele

Sequential modification of two amino acid residues (a histidyl and a cysteinyl residue), both essential for the enzymatic function of bacterial luciferase from Beneckea harveyi, has been conducted to determine if the inactivation arising from the chemical modification of either of these residues is due to a conformational change. This experimental approach has shown that modification of the histidyl or cysteinyl residue did not affect the reactivity of the remaining 'essential' residue, suggesting that chemical modification had not caused a change in conformation. Furthermore, since substrates protect luciferase against inactivation due to modification of either of these residues, it was possible to determine if the initial modification of the histidyl or cysteinyl residue prevented substrate binding by conducting the modification of the remaining residue (i.e., the cysteinyl or histidyl residue, respectively) in the presence of substrates. The results have shown that after modification of the histidyl residue substrates no longer protected the cysteinyl residue against modification, whereas after modification of the cysteinyl residue substrates still protected the histidyl residue against modification. These results have provided evidence that the histidyl residue and not the cysteinyl residue of luciferase is essential for the binding of substrates in the bacterial bioluminescent reaction.     

The active-site arginine of S-adenosylmethionine synthetase orients the reaction intermediate

S-Adenosylmethionine (AdoMet) synthetase catalyzes the formation of AdoMet and tripolyphosphate (PPPi) from ATP and L-methionine and the subsequent hydrolysis of the PPPi to PPi and Pi before product release. Little is known about the roles of active-site residues involved in catalysis of the two sequential reactions that occur at opposite ends of the polyphosphate chain. Crystallographic studies of Escherichia coli AdoMet synthetase showed that arginine-244 is the only arginine near the polyphosphate-binding site. Arginine-244 is embedded as the seventh residue in the conserved sequence DxGxTxxKxI which is also found at the active site of inorganic pyrophosphatases, suggesting a potential pyrophosphate-binding motif. Chemical modification of AdoMet synthetase by the arginine-specific reagents phenylglyoxal or p-hydroxyphenylglyoxal inactivates the enzyme. ATP and PPPi protect the enzyme from inactivation, consistent with the presence of an important arginine residue in the vicinity of the polyphosphate-binding site. Site-specific mutagenesis has been used to change the conserved arginine-244 to either leucine (R244L) or histidine (R244H). In the overall reaction, the R244L mutant has the kcat reduced approximately 10(3)-fold, with a 7 to 10-fold increase in substrate Km values; the R244H mutant has an approximately 10(5)-fold decrease in kcat. In contrast, the kcat values for hydrolysis of added PPPi by the R244L and R244H mutants have been reduced by less than 2 orders of magnitude. In contrast to the wild-type enzyme in which 98% of the Pi formed originates as the gamma-phosphoryl group of ATP, in the R244L mutant the orientation of the PPPi intermediate equilibrates at the active site yielding equal amounts of Pi from the alpha- and gamma-phosphoryl groups of ATP. Thus, the active-site arginine has a profound role in the cleavage of PPPi from ATP during AdoMet formation and in maintaining the orientation of PPPi in the active site, while playing a lesser role in the subsequent PPPi hydrolytic reaction.     

Activation of soluble guanylyl cyclase by the nitrovasodilator 3-morpholinosydnonimine involves formation of S-nitrosoglutathione

Microenvironment and conformation of the active site of xylanase from an extremophilic Bacillus was deciphered for the first time using fluorescence spectroscopy. NBS modified enzyme showed complete inactivation and the kinetic analysis implicated the presence of an essential tryptophan at the active site of xylanase. Xylan (0.5%) protected the enzyme completely from inactivation with NBS, whereas it afforded 35% protection against the loss of fluorescence, suggesting that not all the tryptophans are involved at the substrate binding site. Quenching studies revealed that acrylamide was more efficient than KI and CsCl as indicated by the higher Stern-Volmer quenching constants (Ksv). The steric factor represented by the percentage accessibility of the tryptophan residues of XylII was higher with the positively charged Cs+ (80) than with the negatively charged I- (10), suggesting that the tryptophan residues are located in a relatively electronegative environment. In the presence of 6 M Gdn HCl the fluorescence shifted to 350 nm with increased accessibility of the fluorophore to the quenchers. The proximity of the essential carboxyl groups with a high pKa value of 6.9 [Chauthaiwale and Rao (1994) Biochim. Biophys. Acta] probably contributes to the electronegative environment of the tryptophan residue. Our results on sequence analysis of the gene encoding for XylII (Accession Number U83602 in the GenBank database) have shown that Trp 61 is highly conserved and may play a role in the structure-function relationship of the enzyme.     

Block of brain sodium channels by peptide mimetics of the isoleucine, phenylalanine, and methionine (IFM) motif from the inactivation gate

Inactivation of sodium channels is thought to be mediated by an inactivation gate formed by the intracellular loop connecting domains III and IV. A hydrophobic motif containing the amino acid sequence isoleucine, phenylalanine, and methionine (IFM) is required for the inactivation process. Peptides containing the IFM motif, when applied to the cytoplasmic side of these channels, produce two types of block: fast block, which resembles the inactivation process, and slow, use-dependent block stimulated by strong depolarizing pulses. Fast block by the peptide ac-KIFMK-NH2, measured on sodium channels whose inactivation was slowed by the alpha-scorpion toxin from Leiurus quinquestriatus (LqTx), was reversed with a time constant of 0.9 ms upon repolarization. In contrast, control and LqTx-modified sodium channels were slower to recover from use-dependent block. For fast block, linear peptides of three to six amino acid residues containing the IFM motif and two positive charges were more effective than peptides with one positive charge, whereas uncharged IFM peptides were ineffective. Substitution of the IFM residues in the peptide ac-KIFMK-NH2 with smaller, less hydrophobic residues prevented fast block. The positively charged tripeptide IFM-NH2 did not cause appreciable fast block, but the divalent cation IFM-NH(CH2)2NH2 was as effective as the pentapeptide ac-KIFMK-NH2. The constrained peptide cyclic KIFMK containing two positive charges did not cause fast block. These results indicate that the position of the positive charges is unimportant, but flexibility or conformation of the IFM-containing peptide is important to allow fast block. Slow, use-dependent block was observed with IFM-containing peptides of three to six residues having one or two positive charges, but not with dipeptides or phenylalanine-amide. In contrast to its lack of fast block, cyclic KIFMK was an effective use-dependent blocker. Substitutions of amino acid residues in the tripeptide IFM-NH2 showed that large hydrophobic residues are preferred in all three positions for slow, use-dependent block. However, substitution of the large hydrophobic residue diphenylalanine or the constrained residues phenylglycine or tetrahydroisoquinoline for phe decreased potency, suggesting that this phe residue must be able to enter a restricted hydrophobic pocket during the binding of IFM peptides. Together, the results on fast block and slow, use-dependent block indicate that IFM peptides form two distinct complexes of different stability and structural specificity with receptor site(s) on the sodium channel. It is proposed that fast block represents binding of these peptides to the inactivation gate receptor, while slow, use-dependent block represents deeper binding of the IFM peptides in the pore.     

Cleavage efficiency of the novel aspartic protease yapsin 1 (Yap3p) enhanced for substrates with arginine residues flanking the P1 site: correlation with electronegative active-site pockets predicted by molecular modeling

Yapsin 1, a novel aspartic protease with unique specificity for basic residues, was shown to cleave CCK13-33 at Lys23. Molecular modeling of yapsin 1 identified the active-site cleft to have negative residues close to or within the S6, S3, S2, S1, S1', S2', and S3' pockets and is more electronegative than rhizopuspepsin or endothiapepsin. In particular, the S2' subsite has three negative charges in and close to this pocket that can provide strong electrostatic interactions with a basic residue. The model, therefore, predicts that substrates with a basic residue in the P1 position would be favored with additional basic residues binding to the other electronegative pockets. A deletion of six residues close to the S1 pocket in yapsin 1, relative to rhizopuspepsin and other aspartic proteases of known 3D structure, is likely to affect its specificity. The model was tested using CCK13-33 analogues. We report that yapsin 1 preferentially cleaves a CCK13-33 substrate with a basic residue in the P1 position since the substrates with Ala in P1 were not cleaved. Furthermore, the cleavage efficiency of yapsin 1 was enhanced for CCK13-33 analogues with arginine residues flanking the P1 position. An alanine residue, substituting for the arginine residue in the P6 position in CCK13-33, resulted in a 50% reduction in the cleavage efficiency. Substitution with arginine residues downstream of the cleavage site at the P2', P3', or P6' position increased the cleavage efficiency by 21-, 3- and 7-fold, respectively. Substitution of Lys23 in CCK13-33 with arginine resulted not only in cleavage after the substituted arginine residue, but also forced a cleavage after Met25, suggesting that an arginine residue in the S2' pocket is so favorable that it can affect the primary specificity of yapsin 1. These results are consistent with the predictions from the molecular model of yapsin 1.     

Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase

The free cysteine residues in the extremely thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized using selective chemical modification with the stable nitroxyl biradical bis(1-oxy-2,2,5,5-tetramethyl-3-imidazoline-4-yl)disulfide, via a thiol-disulfide exchange reaction and with 2[14C]iodoacetic acid, via S-alkylation. The respective reactions were monitored by electron paramagenetic resonance (EPR) and by the incorporation of the radioactive label. In native TBADH, the rapid modification of one cysteine residue per subunit by the biradical and the concomitant loss of catalytic activity was reversed by DTT. NADP protected the enzyme from both modification and inactivation by the biradical. RPLC fingerprint analysis of reduced and S-carboxymethylated lysyl peptides from the radioactive alkylated enzyme identified Cys 203 as the readily modified residue. A second cysteine residue was rapidly modified with both modification reagents when the catalytic zinc was removed from the enzyme by o-phenanthroline. This cysteine residue, which could serve as a putative ligand to the active-site zinc atom, was identified as Cys 37 in RPLC. The EPR data suggested a distance of < or 10 A between Cys 37 and Cys 203. Although Cys 283 and Cys 295 were buried within the protein core and were not accessible for chemical modification, the two residues were oxidized to cystine when TBADH was heated at 75 degrees C, forming a disulfide bridge that was not present in the native enzyme, without affecting either enzymatic activity or thermal stability. The status of these cysteine residues was verified by site directed mutagenesis.     

Chemical modification of bacterial luciferase with ethoxyformic anhydride: evidence for an essential histidyl residue

Bacterial luciferase is a heteropolymeric protein (alphabeta) that catalyses the conversion of chemical energy to light by oxidation of a reduced flavin mononucleotide and a long chain aliphatic aldehyde. Elucidation of the specific amino acid residues involved in the enzymatic reaction is essential for understanding the mechanisms of the bioluminescent reaction. Luciferase has been found to be inactivated by ethoxyformic anhydride with a second-order rate constant of 146 M-1 min-1 at pH 6.1 and 0 degrees C with a concomitant increase in absorbance at 240 nm due to formation of ethoxyformylhistidyl derivatives. Activity could be restored by hydroxylamine and the pH curve of inactivation indicated the involvement of a residue having a pKa of 6.8. Both substrates, FMNH2 and aldehyde, protected the enzyme against inactivation, suggesting that the modification occurred at or near the active site. Incorporation of [14C]ethoxyformyl groups in luciferase indicated that inactivation resulted from the modification of about three histidyl residues, one histidine being found on the alpha subunit and two on the beta subunit. Hybridization experiments, in which ethoxyformylluciferase, alphambetam, was complemented with native subunits, alpha or beta, showed that the hybrid alphambetam, has the same activity as alphambetam whereas the activity of the hybrid alphabetam, was close to that of the reconstituted luciferase alphabeta. The results indicate that modification of only one histidyl residue on the alpha subunit inactivates luciferase and suggest that this histidyl residue plays an essential role in the mechanism of the bacterial bioluminescent reaction.     

The responsiveness of generic quality of life instruments in rheumatic diseases. A systematic review of randomized controlled trials

Pseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, is the first example from a prokaryote of unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3(p-nitrophenoxy)propane. To identify the catalytic residue(s) of PCP, chemical modification was carried out using carboxyl residue-specific reagents, carbodiimides. PCP was inactivated effectively by N,N'-dicyclohexylcarbodiimide (DCCD) with pseudo-first-order kinetics. For the inactivation, 0.7 mol DCCD was involved per 1 mol PCP. The effects of pH and methanol on the inactivation showed that two carboxyl residues (Asp and/or Glu) were involved in the reaction. The inactivation by DCCD was prevented by a competitive inhibitor, tyrostatin, or a synthetic substrate in a concentration-dependent manner. Based on these data, differential labeling of PCP with DCCD was carried out: Firstly, PCP was treated with cold DCCD in the presence of tyrostatin. After removal of the tyrostatin, which covered the substrate binding site, by dialysis, the PCP was treated with [14C]DCCD to label carboxyl residue(s) essential for its function. Two labeled peptides were isolated by HPLC from a trypsin digest of cold- and [14C]DCCD modified PCP. On analysis of their amino acid sequences, it was revealed that the [14C]DCCD was bound to Asp140 and Glu222 of PCP, respectively. Based on these data, it was strongly suggested that Asp140 and Glu222 of PCP were involved in its catalytic function or substrate binding.     

Binding characteristics of bovine lactoferrin to the cell surface of Clostridium species and identification of the lactoferrin-binding protein

The binding characteristics of bovine lactoferrin (bLf) to cells of the Clostridium species were observed by using a horseradish peroxidase-bLf conjugate. A bLf-binding protein (BP) having a relative molecular mass of about 33 kDa was confirmed in the surface layer components from 7 strains of the Clostridium species. The binding of the conjugate to bLf-BP or C. perfringens was strongly blocked by intact Lfs, lysine or arginine residues modified bLf, and deglycosylated bLf, but was not by other milk proteins or by the constituent sugars of glycan. Bacterial growth was inhibited by bLf, but was slightly inhibited by lysine residues modified bLf or deglycosylated bLf. Lactoferricin B did not block the binding of the conjugate, but strongly inhibited the bacterial growth. This suggests that the lysine or arginine residues and glycan of bLf hardly participated in binding bLf to the bacterial cells, but that the amino acid residues and glycan played an important role in inhibiting the growth of bacteria.     

Affinity labeling of rat liver carbamyl phosphate synthetase I by 5'-p-fluorosulfonylbenzoyladenosine

The ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA) has been used to study the interaction of MgATP with rat liver carbamyl phosphate synthetase I. Incubation of the enzyme with concentrations of FSBA as low as 0.025 mM produced considerable inactivation (41% at 120 min); identical rates and extents of reaction were produced by 0.5, 1, and 2 mM FSBA. Of the substrates for carbamyl phosphate synthetase I, only MgATP protected against FSBA inactivation. In the presence of a constant concentration of MgATP, increasing the FSBA concentration led to increased inhibition. Conversely, an increase in MgATP concentration led to decreased inhibition from a constant concentration of FSBA. Other nucleotide triphosphates provided no protection against FSBA inactivation. Addition of dithiothreitol to the FSBA-inactivated enzyme led to partial reactivation, suggesting that cysteine residue(s) were involved in the FSBA reaction. 5,5'-Dithiobis(2-nitrobenzoic acid) titration of the free sulfhydryl groups on the enzyme confirmed that cysteine residues were involved in reaction with FSBA; titration of the enzyme after incubation in the absence and presence of FSBA yielded values of 21 and 18(+/- 1), respectively. Binding studies with 5'-p-fluorosulfonylbenzoyl[2-3H]adenosine indicated that: 4 amino acid residues were involved in reaction with FSBA; 2 of these reaction sites were cysteine residues and 2 were noncysteine residues; MgATP protected one of the cysteine residues and one of the noncysteine residues from reaction with FSBA; the MgATP-protected noncysteine residue is essential for fully activity. These data strongly suggest that FSBA is an affinity label for two distinct MgATP sites on carbamyl phosphate synthetase I.     

Oxidative inactivation of gastric peroxidase by site-specific generation of hydroxyl radical and its role in stress-induced gastric ulceration

We have shown earlier that restraint-cold stress-induced gastric ulceration in rats is caused by metal ion-dependent generation of hydroxyl radical (OH.) and oxidative inactivation of the gastric peroxidase (GPO), an important H2O2 scavenging enzyme. To study the mechanism of the oxidative damage of GPO, the purified enzyme was exposed to an OH. generating system containing Cu2+, ascorbate, and H2O2. Kinetic studies indicate that the enzyme is inactivated in a time-dependent process showing saturation with respect to Cu2+ concentration. The enzyme specifically requires Cu2+ and is not inactivated by the same concentration of Fe2+, Mn2+, or Zn2+. Sensitivity to catalase indicates the critical role of H2O2 in the inactivation. Inactivation is insensitive to superoxide dismutase, suggesting no role of superoxide. The rate of inactivation is not increased in D2O excluding the involvement of singlet oxygen in the process. However, OH. scavengers such as benzoate or mannitol cannot prevent inactivation. The results indicate a plausible generation of OH. within the enzyme molecule as the cause of inactivation. Fragmentation of peptide linkage or intramolecular crosslinking, gross change of tertiary structure, or change in intrinsic tryptophan fluorescence which occurs in "global" oxidation are not evident. Inactivation is dependent on pH and from a plot of K(obs) of inactivation against pH, the controlling role of an ionizable group of the enzyme having a pka of 7.8 could be suggested, deprotonation of which favors inactivation. Amino acid analysis shows a specific loss of two lysine residues in the inactivated enzyme. Competitive kinetic studies indicate that pyridoxal phosphate, a specific modifier of the lysine residue, prevents inactivation by competing with Cu2+ for binding at the GPO. A Cu2+ binding motif consisting at least of two lysine residues exists in GPO, which specifically binds Cu2+ and generates OH.. The radical oxidizes the lysine residues and perturbs the heme environment to cause inactivation. We suggest that oxidative damage of GPO is mediated by site-specific generation of OH. and not by the OH. generated in the bulk phase.     

Increased UV exposure in Finland in 1993

N-Ethylmaleimide (NEM) inhibited the H(+)-ATPase (EC 3.6.1.35) from Kluyveromyces lactis with a second-rate constant of 200 M-1 min-1. H(+)-ATPase was partially protected by Mg-ADP. Low concentrations of Mg protected ATPase from the effects of NEM, while high Mg sensitized ATPase to NEM. The reaction of 14C-NEM with the native enzyme modified three cysteine residues/monomer, two of which were involved in 80% of the inactivation of the enzyme. In the presence of Mg-ADP, NEM binding to the first residue had only a slight effect on the activity (10-20% inhibition). After further incubation, the modification of a second cysteine residue (probably cys-221) inactivated the ATPase. Methyl methanethiosulfonate did not inhibit the H(+)-ATPase but resulted in a NEM-resistant H(+)-ATPase. There seems to be at least one cys (probably cys-532) at, or near, the nucleotide binding site of the H(+)-ATPase, which does not appear to be essential for activity. Modification of a second cys residue (cys-221) also resulted in inactivation by NEM; this residue was not protected by ADP and thus probably is not at the ATP binding site.     

Localization of critical histidyl residues required for vinblastine-induced tubulin polymerization and for microtubule assembly

Vinblastine-induced tubulin polymerization is electrostatically regulated and shows pH dependence with a pI approximately 7.0 suggesting the involvement of histidyl residues. Modification of histidyl residues of tubulin with diethylpyrocarbonate (DEPC) at a mole ratio of 0.74 (DEPC/total His residues) for 3 min at 25 degreesC completely inhibited vinblastine-induced polymerization with little effect on microtubule assembly. Under these conditions DEPC reacts only with histidyl residues. For complete inhibition two histidyl residues have to be modified. Demodification of the carboxyethyl histidyl derivatives by hydroxylamine led to nearly complete recovery of polymerization competence. Labeling with [14C]DEPC localized both of these histidyl residues on beta-tubulin at beta227 and beta264. Similarly, tubulin modification with DEPC for longer times (8 min) resulted in complete inhibition of microtubule assembly, at which time approximately 4 histidyl residues had been modified. This inhibition by DEPC was also reversed by hydroxylamine. The third histidyl residue was found on alpha-tubulin at alpha88. Thus, two charged histidyl residues are obligatorily involved in vinblastine-induced polymerization, whereas a different histidyl residue on a different tubulin monomer is involved in microtubule assembly.     

Chemical modification of arginines by 2,3-butanedione and phenylglyoxal causes closure of the mitochondrial permeability transition pore

We have investigated the role of arginine residues in the regulation of the mitochondrial permeability transition pore, a cyclosporin A-sensitive inner membrane channel. Isolated rat liver mitochondria were treated with the arginine-specific chemical reagent 2, 3-butanedione or phenylglyoxal, followed by removal of excess free reagent. After this treatment, mitochondria accumulated Ca2+ normally, but did not undergo permeability transition following depolarization, a condition that normally triggers opening of the permeability transition pore. Inhibition by 2,3-butanedione and phenylglyoxal correlated with matrix pH, suggesting that the relevant arginine(s) are exposed to the matrix aqueous phase. Inhibition by 2,3-butanedione was potentiated by borate and was reversed upon its removal, whereas inhibition by phenylglyoxal was irreversible. Treatment with 2,3-butanedione or phenylglyoxal after induction of the permeability transition by Ca2+ overload resulted in pore closure despite the presence of 0.5 mM Ca2+. At concentrations that were fully effective at inhibiting the permeability transition, these arginine reagents (i) had no effect on the isomerase activity of cyclophilin D and (ii) did not affect the rate of ATP translocation and hydrolysis, as measured by the production of a membrane potential upon ATP addition in the presence of rotenone. We conclude that reaction with 2,3-butanedione and phenylglyoxal results in a stable chemical modification of critical arginine residue(s) located on the matrix side of the inner membrane, which, in turn, strongly favors a closed state of the pore.     

An arginine residue (Arg101), which is conserved in many GroEL homologues, is required for interactions between the two heptameric rings

Homologous recombination was used to construct a series of hybrid chaperonin genes, containing various lengths of Escherichia coli groEL replaced by the equivalent region from the homologous cpn60-1 gene of Rhizobium leguminosarum. Analysis of proteins produced by these hybrids showed that many of them formed structures with properties consistent with their being single heptameric rings under some conditions, as opposed to the double ring form in which both the GroEL and the Cpn60-1 proteins are found. By determining precise cross-over points, two regions in Cpn60-1 were defined which appeared to be critical for ring-ring interactions. Within one of these regions is a highly conserved arginine residue (Arg101), which we hypothesised to interact with a residue or residues toward the C terminus of the protein, this contact being required for double rings to form. To test this hypothesis, we mutagenised this residue from arginine to threonine in chaperonin genes from two different species of Rhizobium. In both cases, proteins which ran on non-denaturing gels as single rings were produced. Conversion of Arg101 to serine also had the same effect, whereas conversion of Arg101 to lysine did not. Two different single rings created by homologous recombination could be converted back to double rings by changing the threonine, which naturally occurs at this position in E. coli GroEL, back to arginine. The in vivo properties of the proteins were investigated by complementation following deletion of the chromosomal copy of the groEL gene, and by monitoring the ability of cells expressing the hybrid proteins to plate bacteriophage. Most of the hybrid and mutant proteins were functional in these assays, despite their altered properties compared to wild-type GroEL.     

Identification of a cysteine residue essential for activity of protein farnesyltransferase. Cys299 is exposed only upon removal of zinc from the enzyme

Protein farnesyltransferase (FTase) is a zinc metalloenzyme that performs a post-translational modification on many proteins that is critical for their function. The importance of cysteine residues in FTase activity was investigated using cysteine-specific reagents. Zinc-depleted FTase (apo-FTase), but not the holoenzyme, was completely inactivated by treatment with N-ethylmaleimide (NEM). Similar effects were detected after treatment of the enzyme with iodoacetamide. The addition of zinc to apo-FTase protects it from inactivation by NEM. These findings indicated the presence of specific cysteine residue(s), potentially located at the zinc binding site, that are required for FTase activity. We performed a selective labeling strategy whereby the cysteine residues exposed upon removal of zinc from the enzyme were modified with [3H]NEM. The enzyme so modified was digested with trypsin, and four labeled peptides were identified and sequenced, one peptide being the major site of labeling and the remaining three labeled to lesser extents. The major labeled peptide contained a radiolabeled cysteine residue, Cys299, that is in the beta subunit of FTase and is conserved in all known protein prenyltransferases. This cysteine residue was changed to both alanine and serine by site-directed mutagenesis, and the mutant proteins were produced in Escherichia coli and purified. While both mutant proteins retained the ability to bind farnesyl diphosphate, they were found to have lost essentially all catalytic activity and ability to bind zinc. These results indicate that the Cys299 in the beta subunit of FTase plays a critical role in catalysis by the enzyme and is likely to be one of the residues that directly coordinate the zinc atom in this enzyme.     

Three residues predicted by molecular modeling to interact with the purine moiety alter ligand binding and channel gating in cyclic nucleotide-gated channels

Cytoplasmic cAMP and cGMP are soluble cellular messengers that directly activate cyclic nucleotide-gated (CNG) channels. These channels mediate sensory transduction in photoreceptors and olfactory neurons. The closely related CNG channels in these cell types have different nucleotide activation profiles, and we have investigated the molecular basis of their nucleotide selectivity properties. Previously, we predicted that the purine moiety of the nucleotide interacts with residues F533, K596, and D604 (bovine rod alpha CNG channel subunit sequences) of the nucleotide binding domain. In this study, we replaced these three residues with the corresponding residues of the bovine olfactory CNG channel. Mutations at each position altered the nucleotide activation of the rod CNG channels. In a mutant where K596 was replaced with arginine, cAMP-activated currents were enhanced 8-12-fold, suggesting that residue 596 influences channel gating. Thermodynamic cycle analysis of the data showed that (1) the residues are energetically coupled and (2) energetic coupling exists between the potentiating effects of Ni2+ and the replacement of F533 with tyrosine. These data suggest that changes in one of the residues alter the purine contacts with the other residues and that F533 communicates with the C-linker region of the channel involved in Ni2+ potentiation.