Identification of phenolyl cobamide from the homoacetogenic bacterium Sporomusa ovata

Phenolyl cobamide was isolated from cyanide extractions of the anaerobic eubacterium Sporomusa ovata. The proposed corrinoid structure [Co alpha,Co beta-(monocyano,monoaquo)-phenolyl cobamide] has been deduced from 1H NMR, fast-atom-bombardment mass spectroscopy and ultraviolet/visible spectroscopy data. The complete corrinoid resembled p-cresolyl cobamide [Co alpha,Co beta-(monocyano,monoaquo)-p-cresolyl cobamide], which recently has been obtained from cyanide extractions of the same bacterium. The structures and chemical properties of both cobamides with uncoordinated nucleotides differed significantly from those of vitamin B12 [Co alpha-[alpha-(5,6-dimethylbenzimidazolyl)]-Co beta-cyanocobamide]. Sporomusa synthesized coenzymes of phenolyl cobamide and p-cresolyl cobamide in considerable amounts of 400 nmol/g and 1700 nmol/g dry cells, respectively. More than 90% of the complete corrinoid pool of the homoacetogenic bacterium consisted of these two corrinoids, indicating that they are physiologically important coenzymes of the bacterial metabolism.     

Evidence for a functionally important histidine residue in human tyrosine hydroxylase

Summary Recombinant human tyrosine hydroxylase isozyme 1 (hTH1) shows a time- and concentration-dependent loss of catalytic activity when incubated with diethylpyrocarbonate (DEP) after reconstitution with Fe(II). The inactivation follows pseudo-first order kinetics with a second order rate constant of 300 M^–1 min^–1 at pH 6.8 and 20°C and is partially reversed by hydroxylamine. The difference absorption spectrum of the DEP-modified vs native enzyme shows a peak at 244 nm, characteristic of mono-N-carbethoxy-histidine. Up to five histidine residues are modified per enzyme subunit by a five-fold excess of the reagent, and two of them are protected from inactivation by the active site inhibitor dopamine. However, derivatization of only one residue appears to be responsible for the inactivation. Thus, no inactivation by DEP was found when the apoenzyme was preincubated with this reagent prior to its reconstitution with Fe(II), modifying four histidine residues.Abbreviations BH₄ (6R)-l-erythro-tetrahydrobiopterin - DEP diethylpyrocarbonate - DOPA 3,4-dihydroxyphenylalanine - hTH1 human tyrosine hydroxylase isoenzyme 1 - apo-hTH1 apoenzyme of hTH1 - Fe(II)-hTH1 holoenzyme (iron reconstituted) of hTH1 - dopamine-Fe(III)-hTH1 holoenzyme of hTH1 with dopamine bound - TH tyrosine hydroxylase     

Evidence for an essential histidine residue in the ascorbate-binding site of cytochrome b561

Cytochrome b(561) mediates equilibration of the ascorbate/semidehydroascorbate redox couple across the membranes of secretory vesicles. The cytochrome is reduced by ascorbic acid and oxidized by semidehydroascorbate on either side of the membrane. Treatment with diethyl pyrocarbonate (DEPC) inhibits reduction of the cytochrome by ascorbate, but this activity can be restored by subsequent treatment with hydroxylamine, suggesting the involvement of an essential histidine residue. Moreover, DEPC inactivates cytochrome b(561) more rapidly at alkaline pH, consistent with modification of a histidine residue. DEPC does not affect the absorption spectrum of cytochrome b(561) nor does it change the midpoint reduction potential, confirming that histidine modification does not affect the heme. Ascorbate protects the cytochrome from inactivation by DEPC, indicating that the essential histidine is in the ascorbate-binding site. Further evidence for this is that DEPC treatment inhibits oxidation of the cytochrome by semidehydroascorbate but not by ferricyanide. This supports a reaction mechanism in which ascorbate loses a hydrogen atom by donating a proton to histidine and transferring an electron to the heme.     

Dichloromethane as the sole carbon source for an acetogenic mixed culture and isolation of a fermentative, dichloromethane-degrading bacterium.

Dichloromethane (DCM) is utilized by the strictly anaerobic, acetogenic mixed culture DM as a sole source of carbon and energy for growth. Growth with DCM was linear, and cell suspensions of the culture degraded DCM with a specific activity of 0.47 mkat/kg of protein. A mass balance of 2 mol of chloride and 0.42 mol of acetate per mol of DCM was observed. The dehalogenation reaction showed similar specific activities under both anaerobic and aerobic conditions. Radioactivity from [14C]DCM in cell suspensions was recovered largely as 14CO2 (58%), [14C]acetate (23%), and [14C]formate (11%), which subsequently disappeared. This suggested that formate is a major intermediate in the pathway from DCM to acetate. Efforts to isolate from culture DM a pure culture capable of anaerobic growth with DCM were unsuccessful, although overall acetogenesis and the partial reactions are thermodynamically favorable. We then isolated bacterial strains DMA, a strictly anaerobic, gram-positive, endospore-forming rod, and DMB, a strictly anaerobic, gram-negative, endospore-forming homoacetogen, from culture DM. Both strain DMB and Methanospirillum hungatei utilized formate as a source of carbon and energy. Coculture of strain DMA with either M. hungatei or strain DMB in solid medium with DCM as the sole added source of carbon and energy was observed. These data support a tentative scheme for the acetogenic fermentation of DCM involving interspecies formate transfer from strain DMA to the acetogenic bacterium DMB or to the methanogen M. hungatei.     

Evidence for an essential histidine residue in the Neurospora crassa plasma membrane H+-ATPase

The Neurospora crassa plasma membrane H+-ATPase is rapidly inactivated in the presence of diethyl pyrocarbonate (DEP). The reaction is pseudo-first-order showing time- and concentration-dependent inactivation with a second-order rate constant of 385-420 M-1.min-1 at pH 6.9 and 25 degrees C. The difference spectrum of the native and modified enzyme has a maximum near 240 nm, characteristic of N-carbethoxyhistidine. No change in the absorbance of the inhibited ATPase at 278 nm or in the number of modifiable sulfhydryl groups is observed, indicating that the inhibition is not due to tyrosine or cysteine modification, and the inhibition is irreversible, ruling out serine residues. Furthermore, pretreatment of the ATPase with pyridoxal phosphate/NaBH4 under the conditions of the DEP treatment does not inhibit the ATPase and does not alter the DEP inhibition kinetics, indicating that the inactivation by DEP is not due to amino group modification. The pH dependence of the inactivation reaction indicates that the essential residue has a pKa near 7.5, and the activity lost as a result of H+-ATPase modification by DEP is partially recovered after hydroxylamine treatment at 4 degrees C. Taken together, these results strongly indicate that the inactivation of the H+-ATPase by DEP involves histidine modification. Analyses of the inhibition kinetics and the stoichiometry of modification indicate that among eight histidines modified per enzyme molecule, only one is essential for H+-ATPase activity. Finally, ADP protects against inactivation by DEP, indicating that the essential residue modified may be located at or near the nucleotide binding site.     

Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases.

(4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes.     

Evidence for dynamics in proteins as a mechanism for ligand dissociation

Signal transduction, regulatory processes and pharmaceutical responses are highly dependent upon ligand residence times. Gaining insight into how physical factors influence residence times (1/k(off)) should enhance our ability to manipulate biological interactions. We report experiments that yield structural insight into k(off) involving a series of eight 2,4-diaminopyrimidine inhibitors of dihydrofolate reductase whose binding affinities vary by six orders of magnitude. NMR relaxation-dispersion experiments revealed a common set of residues near the binding site that undergo a concerted millisecond-timescale switching event to a previously unidentified conformation. The rate of switching from ground to excited conformations correlates exponentially with the binding affinity K(i) and k(off), suggesting that protein dynamics serves as a mechanical initiator of ligand dissociation within this series and potentially for other macromolecule-ligand systems. Although the forward rate of conformational exchange, k(conf,forward), is faster than k(off), the use of the ligand series allowed for connections to be drawn between kinetic events on different timescales.     

Chemical modification of chloroperoxidase with diethylpyrocarbonate. Evidence for the presence of an essential histidine residue

Chloroperoxidase from Caldariomyces fumago is well documented as an extremely versatile catalyst, and studies are currently being conducted to delineate the fine structural features that allow the enzyme to possess chemical and physical similarities to the peroxidases, catalases, and P-450 cytochromes. Earlier investigations of ligand binding to the heme iron of chloroperoxidase, along with the presence of an invariant distal histidine residue in the active site of peroxidases and catalases, have led to the hypothesis that chloroperoxidase also possesses an essential histidine residue that may participate in catalysis. To address this in a more direct fashion, chemical modification studies were initiated with diethylpyrocarbonate. Incubation of chloroperoxidase with this reagent resulted in a time-dependent inactivation of enzyme. Kinetic analysis revealed that the inactivation was due to a simple bimolecular reaction. The rate of inactivation exhibited a pH dependence, indicating that modification of a titratable residue with a pKa value of 6.91 was responsible for inactivation; this data provided strong evidence for histidine derivatization by diethylpyrocarbonate. To further support these results, inactivation due to cysteine, tyrosine, or lysine modification was ruled out. The stoichiometry of histidine modification was estimated by the increase in absorption at 246 nm, and it was found that more than 1 histidine residue was derivatized when chloroperoxidase was inactivated with diethylpyrocarbonate. However, it was shown that the rates of modification and inactivation were not equivalent. This was interpreted to reflect that both essential and nonessential histidine residues were modified by diethylpyrocarbonate. Kinetic analysis indicated that modification of a single essential histidine residue was responsible for inactivation of the enzyme. Studies with [14C]diethylpyrocarbonate provided stoichiometric support that derivatization of a single histidine inactivated chloroperoxidase. Based on sequence homology with cytochrome c peroxidase, histidine 38 was identified as a likely candidate for the distal residue. Molecular modeling, based on secondary structure predictions, allows for the construction of an active site peptide, and implicates a number of other residues that may participate in catalysis.     

Evidence for the in vivo deamidation and isomerization of an asparaginyl residue in cytosolic serine hydroxymethyltransferase

Rabbit liver cytosolic serine hydroxymethyltransferase exists in several subforms which have different isoelectric points. Incubation of the purified enzyme with chymotrypsin cleaves the enzyme at Trp14. The released amino-terminal 14-mer peptide was shown to exist in three forms of equal concentration. The peptides differ in structure only at the asparaginyl residue at position 5. In addition to asparagine at this position we found both aspartyl and isoaspartyl residues. The deamidation of Asn5 does not appear to occur during the purification of the enzyme. The in vitro rate of deamidation of Asn5 in the enzyme is more than 5-fold slower than the rate of deamidation of this residue in the free 14-mer peptide. The isoaspartyl residue at position 5 serves as a substrate for protein carboxyl methyltransferase both in the free 14-mer peptide and the native enzyme. The enzyme which has had the amino-terminal 14 residues removed by digestion with chymotrypsin still exists in several forms with different isoelectric points. Reaction of peptides from this enzyme with carboxyl methyltransferase suggests that there is at least one more asparaginyl residue in this enzyme other than Asn5 which has undergone deamidation with the formation of isoaspartyl bonds.     

Evidence for the involvement and role of a corrinoid enzyme in methane formation from acetate in Methanosarcina barkeri

Methane formation from acetate in cell suspensions of Methanosarcina barkeri was inhibited by low concentrations (5 M) of propyl iodide. Inhibition was abolished by short exposure of the suspension to light which strongly indicates that a corrinoid enzyme is involved in methanogenesis from acetate. Propyl iodide (5M) had no effect on the exchange reaction between the carboxyl group of acetate and ¹⁴CO₂, and on methane formation from methanol, from H₂ and methanol, or from H₂ and CO₂. These findings indicate that the proposed corrinoid enzyme has a role in methyl group transfer to coenzyme M after C-C cleavage of acetate.Dedicated to Professor N. Pfennig on the occasion of his 60th birthday     

Evidence for the presence of a critical histidine residue at the active site in glyceraldehyde-3-phosphate dehydrogenase of Ehrlich ascites carcinoma cells

Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (GA3PD) (EC. 1.2.1.12) was completely inactivated by diethyl pyrocarbonate (DEPC), a fairly specific reagent for histidine residues in the pH range of 6.0-7.5. The rate of inactivation was dependent on pH and followed pseudo-first order reaction kinetics. The difference spectrum of the inactivated and native enzymes showed an increase in the absorption maximum at 242 nm, indicating the modification of histidine residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of one essential histidine residue to be responsible for loss of the catalytic activity of EAC cell GA3PD. DEPC inactivation was protected by substrates, D-glyceraldehyde-3-phosphate and NAD, indicating the presence of essential histidine residue at the substrate-binding region of the active site. Double inhibition studies also provide evidence for the presence of histidine residue at the active site.     

Evidence for a spectrin-like protein as a major component of the synaptosomal membrane cytoskeleton

After treatment of synaptosomes with Nonidet-containing buffers, a proportion of the proteins remained insoluble. The major component (50%) of the residue was identified as a spectrin-like protein by immunodetection after mono- and bi-dimensional gel electrophoresis and transfer to nitrocellulose paper. Actin was also present.     

Modification of uridine phosphorylase from Escherichia coli by diethyl pyrocarbonate. Evidence for a histidine residue in the active site of the enzyme.

Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the inactivated enzyme restores the activity. Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate. Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity. The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues. Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification. Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.     

Evidence for histidine in the active site of papain

Papain was irreversibly inhibited by 1,3-dibromoacetone, a reagent designed to react first with the active-site cysteine residue and subsequently with a second nucleophile. The molecular weight of the inhibited enzyme was indistinguishable from that of papain itself, and no evidence of dimeric or oligomeric species was found. The optical-rotatory-dispersion curves of chloroacetone-inhibited papain and 1,3-dibromoacetone-inhibited papain were essentially similar. Amino acid analysis of the 1,3-dibromo[2-¹⁴C]acetone-inhibited enzyme and the performic acid-oxidized material clearly showed that a cysteine and histidine residue had been alkylated through the thiol and N-1 of the imidazole group respectively. These groups must therefore be within 5å of each other in the tertiary structure of papain. Possible mechanistic implications are briefly discussed.     

Nucleic acids from the host bacterium as a major source of nucleotides for three marine bacteriophages

Abstract The incorporation of ³²P-phosphorus into marine bacteriophage nucleic acid was studied in culture experiments to investigate the source of nucleotides used by the phage. We consistently found that the ³²P-specific activity in the phage genome increased during the 11 h incubation and was low relative to the specific activity in the medium, averaging 21% (±SD 5.9) for the three phage isolates. This was in accordance with a mathematical model where most of the nucleotides for phage DNA synthesis were derived from the host cell nucleic acid rather than de novo synthesis. We propose that this metabolic strategy may be common among marine phages, as an adaptation to a nutrient poor environment. Consequently, the contribution of free DNA to the dissolved fraction through phage lysis of bacteria, may be less that previously thought. Also during radiolabelling of bacteriophages in natural water samples, isotope dilution may be dependent on the specific growth rate of the bacterial host.     

Evidence for histidine in the active sites of ficin and stem-bromelain

1. Ficin and stem-bromelain are irreversibly inhibited by 1,3-dibromoacetone, a reagent designed to react first with the active-site cysteine residue and subsequently with a second nucleophile. Evidence is presented that establishes that a histidine residue is within a 5A locus of the active-site cysteine residue in both enzymes. The histidine residue in both enzymes is alkylated at N-1 by dibromoacetone. It is suggested that, as with papain, the thiol and imidazole groups act in concert in the hydrolysis of substrates by these enzymes. 2. The inhibition of thiol-subtilisin with 1,3-dibromoacetone is shown to be due to the alkylation of a cysteine residue only.     

Evidence for histidine in the triethyltin-binding site of rat haemoglobin

One molecule of rat haemoglobin binds two molecules of triethyltin. The binding sites are located on the globin and there is co-operativity between the sites such that the intrinsic affinity constant at pH8.0 increases from 3.5x10(5)m(-1) for the binding of the first triethyltin molecule to 5.0x10(5)m(-1) for the binding of the second. Evidence is presented, from pH studies and the kinetics of inhibition due to photo-oxidation, that each binding site contains two histidine residues.