Reactivity of histidine and lysine side-chains with diethylpyrocarbonate -- a method to identify surface exposed residues in proteins

The chemical modification of amino acid side-chains followed by mass spectrometric detection can reveal at least partial information about the 3-D structure of proteins. In this work we tested diethylpyrocarbonate, as a common histidyl modification agent, for this purpose. Appropriate conditions for the reaction and detection of modified amino acids were developed using angiotensin II as a model peptide. We studied the modification of several model proteins with a known spatial arrangement (insulin, cytochrome c, lysozyme and human serum albumin). Our results revealed that the surface accessibility of residues is a necessary, although in itself insufficient, condition for their reactivity; the microenvironment of side-chains and the dynamics of protein structure also affect the ability of residues to react. However the detection of modified residues can be taken as proof of their surface accessibility, and of direct contact with solvent molecules.     

Diethylpyrocarbonate—An effective agent for the sterilization of different types of nutrient media

A simple and convenient method has been tested for the steriltzation of nutrient media for long-term cultivation of plant cells. Diethylpyrocarbonate is suitable for this task in concentrations about 1000 mg l^-1 The cells cultivated for 15 subsequent passages on media treated by DPC had the same growth parameters, production pattern and ability to transform exogeneous organic compounds as did the controls. The method is suitable for the preparatian of both liquid and agar media, for stabilization of stock solutions and for sterilization of cultivation vessels and tubing.Abbreviations DPC diethylpyrocarbonate - medium MS nutrient medium according to Murashige and Skoog - NAA naphtaleneacetic acid     

Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue

Vacuolar proton pumping pyrophosphatase (H⁺-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PP_i hydrolysis. A histidine-specific modifier, diethylpyrocarbonate (DEPC), could substantially inhibit enzymic activity and H⁺-translocation of vacuolar H⁺-PPase in a concentration-dependent manner. Absorbance of vacuolar H⁺-PPase at 240 nm was increased upon incubation with DEPC, demonstrating that an N-carbethoxyhistidine moiety was probably formed. On the other hand, hydroxylamine, a reagent that can deacylate N-carbethoxyhistidine, could reverse the absorption change at 240 nm and partially restore PP_i hydrolysis activity as well. The pK _a of modified residues of the enzyme was determined to be 6.4, a value close to that of histidine. Thus, we speculate that inhibition of vacuolar H⁺-PPase by DEPC possibly could be attributed to the modification of histidyl residues on the enzyme. Furthermore, inhibition of vacuolar H⁺-PPase by DEPC follows pseudo-first-order rate kinetics. A reaction order of 0.85 was calculated from a double logarithmic plot of the apparent reaction constant against DEPC concentration, suggesting that the modification of one single histidine residue on the enzyme suffices to inhibit vacuolar H⁺-PPase. Inhibition of vacuolar H⁺-PPase by DEPC changes V _max but not K _m values. Moreover, DEPC inhibition of vacuolar H⁺-PPase could be substantially protected against by its physiological substrate, Mg²⁺-PP_i. These results indicated that DEPC specifically competes with the substrate at the active site and the DEPC-labeled histidine residue might locate in or near the catalytic domain of the enzyme. Besides, pretreatment of the enzyme with N-ethylmaleimide decreased the degree of subsequent labeling of H⁺-PPase by DEPC. Taken together, we suggest that vacuolar H⁺-PPase likely contains a substrate-protectable histidine residue contributing to the inhibition of its activity by DEPC, and this histidine residue may located in a domain sensitive to the modification of Cys-629 by NEM.     

Effect of chemical modification of histidines on the copper-induced oligomerization of jack bean urease (EC 3.5.1.5)

Aggregation of jack bean urease (JBU) is associated with alterations of its biological properties, notably the ureolytic and entomotoxic activities. We investigated the influence of metals on protein oligomerization and biological properties. Besides protein aggregation, Cu(2+) induces inhibition of both ureolytic and insecticidal activities of JBU. Chemical modification of histidine residues in JBU with diethylpyrocarbonate (DEPC) decreases its affinity for Cu(2+) and inhibits oligomerization induced by this metal. Furthermore, this modification protects the insecticidal properties of JBU from being inactivated by Cu(2+). Although DEPC-treated JBU displayed lower ureolytic activity, the modified protein is less susceptible to inhibition by Cu(2+) when compared to native enzyme. Our findings show that Cu(2+) promotes JBU aggregation and differently of other heavy metals studied here, it apparently inhibits the ureolytic activity by inducing protein polymerization along with blockage of sulfhydryl groups.     

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     

Identification of histidyl and cysteinyl residues essential for catalysis by 5'-nucleotidase

Inactivation of both cytosolic 5'-nucleotidase and ecto-5'-nucleotidase by diethylpyrocarbonate indicated the presence of an essential histidyl residue which in the cytosolic enzyme was conclusively located at the active site. Inactivation by thiol reagents indicated the presence of an essential cysteinyl residue in both enzymes. The data suggest that both 5'-nucleotidases belong to a group of histidine phosphatases which also includes glucose-6-phosphatase and acid phosphatase. A working hypothesis for the catalytic mechanism of these enzymes is proposed.