Expression, purification, characterization, and NMR studies of highly deuterated recombinant cytochrome c peroxidase.

Two forms of extensively deuterated S. cerevisiae cytochrome c peroxidase (CcP; EC 1.11.1.5) have been overexpressed in E. coli by growth in highly deuterated medium. One of these ferriheme enzyme forms (recDCcP) was produced using >97% deuterated growth medium and was determined to be approximately 84% deuterated. The second form [recD(His)CcP] was grown in the same highly deuterated medium that had been supplemented with excess histidine (at natural hydrogen isotope abundance) and was also approximately 84% deuterated. This resulted in direct histidine incorporation without isotope scrambling. Both of these enzymes along with the corresponding recombinant native CcP (recNATCcP), which was expressed in a standard medium with normal hydrogen isotope abundance, consisted of 294 amino acid polypeptide chains having the identical sequence to the yeast-isolated enzyme, without any N-terminal modifications. Comparative characterizations of all three enzymes have been carried out for the resting-state, high-spin forms and in the cyanide-ligated, low-spin forms. The primary physical methods employed were electrophoresis, UV-visible spectroscopy, hydrogen peroxide reaction kinetics, mass spectrometry, and (1)H NMR spectroscopy. The results indicate that high-level deuteration does not significantly alter CcP's reactivity or spectroscopy. As an example of potential NMR uses, recDCcPCN and recD(His)CcPCN have been used to achieve complete, unambiguous, stereospecific (1)H resonance assignments for the heme hyperfine-shifted protons, which also allows the heme side chain conformations to be assessed. Assigning these important active-site protons has been an elusive goal since the first NMR spectra on this enzyme were reported 18 years ago, due to a combination of the enzyme's comparatively large size, paramagnetism, and limited thermal stability.

Yeast cytochrome c peroxidase expression in Escherichia coli and rapid isolation of various highly pure holoenzymes.

A more efficient 2-day isolation and purification method for recombinant yeast cytochrome c peroxidase produced in Escherichia coli is presented. Two types of recombinant "wild-type" CcP have been produced and characterized, the recombinant nuclear gene sequence and the 294-amino-acid original protein sequence. These two sequences constitute the majority of the recombinant "native" or wild-type CcP currently in production and from which all recombinant variants now derive. The enzymes have been subjected to extensive physical characterizations, including sequencing, UV-visible spectroscopy, HPLC, gel electrophoresis, kinetic measurements, NMR spectroscopy, and mass spectrometry. Less extensive characterization data are also presented for recombinant, perdeuterated CcP, an enzyme produced in >95% deuterated medium. All of these results indicate that the purified recombinant wild-type enzymes are functionally and spectroscopically identical to the native, yeast-isolated wild-type enzyme. This improved method uses standard chromatography to produce highly purified holoenzyme in a more efficient manner than previously achieved. Two methods for assembling the holoenzyme are described. In one, exogenous heme is added at lysis, while in the other heme biosynthesis is stimulated in E. coli. A primary reason for developing this method has been the need to minimize loss of precious, isotope-labeled enzyme and, so, this method has also been used to produce both the perdeuterated and the (15)N-labeled enzyme, as well as several variants.

Improvement of peroxygenase activity by relocation of a catalytic histidine within the active site of horseradish peroxidase.

To examine the role of Arg38 in the peroxidative and peroxygenative activity of horseradish peroxidase (HRP), we have expressed the R38A, R38H, and R38H/H42V mutants. The R38A HRP mutant gives a normal compound I species with H2O2 that is reduced by ferrocyanide to the ferric state without the detectable formation of a compound II species. In the case of the R38H and R38H/H42V mutants, compound I itself is only detected by stopped flow methods. The rates of compound I formation at 4 degrees C are 8.0 x 10(4), 1.3 x 10(6), and 1.6 x 10(3) M-1 s-1 for the R38A, R38H, and R38H/H42V mutants, respectively. The R38A, R38H, and R38H/H42V mutants oxidize guaiacol 10-, 2-, and 55-fold, respectively, more slowly than the wild-type enzyme and oxidize ABTS 6-, 3-, and 32-fold more slowly than the wild-type enzyme. The apparent kcat/K(m) values for thioanisole sulfoxidation and styrene epoxidation indicate that the reaction efficiencies of the R38H and wild-type enzymes are comparable. However, the R38A and R38H/H42V mutants are 190- and 1400-fold more efficient as sulfoxidation catalysts, and 25- and 26-fold more efficient as styrene epoxidation catalysts, respectively, than the wild-type enzyme. Thus, even though Arg38 plays a role in the formation and stabilization of compounds I and II, its replacement by other residues can be used to improve peroxygenative catalysis.

Horseradish peroxidase: partial rescue of the His-42 --> Ala mutant by a concurrent Asn-70 --> Asp mutation.

In horseradish peroxidase (HRP), hydrogen bonding of Asn-70 to His-42 enhances the catalytic activity of the histidine, and mutation of His-42 to a neutral residue greatly decreases peroxidase activity. The N70D/H42A HRP mutant is compared here to the previously characterized H42A mutant to determine if the Asp-70 substitution can rescue the catalytic activity. The N70D/H42A and H42A mutants give Compound I species with a high ratio of H2O2 at the low rates of 37 and 81 M-1 s-1 at 4 degrees C, respectively. The kcat values for the oxidation of guaiacol and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by N70D/H42A HRP are 2.7 and 143 s-1, respectively, compared to values of 0.015 and 0.41 s-1 for the H42A mutant. The kcat values for thioanisole sulfoxidation by the N70D/H42A and H42A mutants are 0.18 and 0.03 s-1, respectively, and the corresponding values for styrene epoxidation are 0.005 and 0.007 s-1. Due to changes in the substrate Km values, the efficiencies of the N70D/H42A and H42A mutants defined by kcat/Km are guaiacol, 5 vs 4; ABTS, 286 vs 68; thioanisole, 3 vs 0.1; and styrene, 0.025 vs 0.002, respectively. The N70D mutation in N70D/H42A HRP thus increases the activity versus the H42A mutant with respect to all four substrates. The increased efficiency is due to enhancements in catalytic steps other than the formation of Compound I.

Rescue of His-42 --> Ala horseradish peroxidase by a Phe-41 --> His mutation. Engineering of a surrogate catalytic histidine.

Formation of the ferryl (FeIV=O) porphyrin radical cation known as Compound I in the reaction of horseradish peroxidase (HRP) with H2O2 is catalyzed by His-42, a residue that facilitates the binding of H2O2 to the iron and subsequent rupture of the dioxygen bond. An H42A mutation was shown earlier to decrease the rate of Compound I formation by a factor of approximately 10(6) and of guaiacol oxidation by a factor of approximately 10(4). In contrast, an F41A mutation has little effect on peroxidative catalysis (Newmyer, S. L., and Ortiz de Montellano, P. R. (1995) J. Biol. Chem. 270, 19430-19438). We report here construction, expression, and characterization of the F41H/H42A double mutant. The pH profile for guaiacol oxidation by this double mutant has a broad maximum at approximately pH 6.3. Addition of H2O2 produces a Compound I species (lambdamax = 406 nm) that is reduced by 1 eq of K4Fe(CN)6 to the ferric state (lambdamax = 407 nm) without the detectable formation of Compound II. A fraction of the heme chromophore is lost in the process. The rate of Compound I formation for the F41H/H42A double mutant is 3.0 x 10(4) M-1 s-1. This is to be compared with 0.9 x 10(7) M-1 s-1 for wild-type HRP and 19 M-1 s-1 for the H42A mutant. The kcat values for guaiacol oxidation by wild-type, H42A, and F41H/H42A HRP are 300, 0.015, and 1.8 s-1. The corresponding kcat values for ABTS oxidation are 4900, 0.41, and 100 s-1, respectively. These results show that a histidine at position 41 substitutes, albeit imperfectly, for His-42 in peroxidative turnover of the enzyme. The F41H/H42A double mutant has peroxidative properties intermediate between those of the native enzyme and the H42A mutant. The F41H/H42A double mutant, however, is a considerably better thioanisole sulfoxidation and styrene epoxidation catalyst than native or H42A HRP. The surrogate catalytic residue introduced by the F41H mutation thus partially compensates for the H42A substitution used to increase access to the ferryl oxygen.

[Equilibrium and kinetic parameters of the interaction of strophanthin K and its peroxidase conjugates with soluble and immobilized specific antibodies]. / Ravnovesnye i kineticheskie parametry vzaimodeistviia strofantina K i ego pereoksidaznykh kon''iugatov s rastvorennymi i immobilizovannymi spetsificheskimi antitelami.

Using solid phase enzyme-linked immunoassay (ELISA), rate constants for the formation and dissociation as well as equilibrium dissociation constants for strophanthin complexes interaction with specific antibodies (both soluble and immobilized on activated polystyrene beads and polyamide membranes) have been determined. A satisfactory correlation was found between dissociation constants for immune complexes determined by ELISA (both soluble and immobilized ones) and associations constants for these complexes determined by the Scatchard method. The experimental values of kinetic and equilibrium constants for strophanthin K and its peroxidase complexes interaction reflect the difference in the nature of interacting particles and their environment. Strophanthin complexes with antibodies appeared to be more stable when formed on adsorbents (in comparison with those formed in solution). Strophanthin interaction with immobilized antibodies occurred at a slower rate than that with soluble antibodies. Strophanthin reacts with antibodies more readily than its complexes both in solution and on solid matrices of various polymeric nature.

[Noncovalent immobilization of catalase on antibodies adsorbed on carbon fabric]. / Nekovalentnaia immobilizatsiia katalazy na antitelakh sorbirovannykh ugol'noi tkan'iu.

Catalase was immobilized on an immunosorbent prepared by anticatalase adsorption on an activated carbon fabric (ACF), and its kinetic parameters were determined. The immobilized catalase activity depended on the binding capacity of anticatalase. Under the optimum conditions (6 micrograms/mg anticatalase, 5.24 nM catalase) the immobilized catalase activity was 1.5-ford higher as compared to soluble catalase. Antibodies stabilized soluble catalase, but decreased its thermostability on immobilization of immunocomplexes on ACF. Noncovalent immobilization of catalase on adsorbed antibodies opens up the way to the use of this approach for immobilization of other oligomeric enzymes.

[The effect of monoclonal and polyclonal antibodies to peroxidase on combined peroxidase oxidation of 4-iodophenol with luminol and 4-aminoantipyrine]. / Vliianie monoklonal'nykh i poliklonal'nykh antitel k peroksidaze na sopriazhennoe pereoksidaznoe okislenie 4-iodfenola s liuminolom i 4-aminoantipirinom.

The kinetics of peroxidase-dependent cooxidation for two substrate pairs [p-iodophenol + 4-aminoantipyrine (AAP) and p-iodophenol + luminol was studied both in the absence and presence of polyclonal antibodies (polyAB), three types of peroxidase-specific monoclonal antibodies (monoAB) and their double or triple mixtures in a wide range of H2O2 concentrations (0.01-10.0 mM). MonoAB 2C, 3E and 9D at concentrations of 0.05-500 nM inhibited the cooxidation of p-iodophenol + AAP at H2O2 concentration above 1.0 mM but activated the cooxidation of p-iodophenol + luminol. The double and triple mixtures of monoAB activated the cooxidation of p-iodophenol + AAP at the same H2O2 concentrations without any effect on the p-iodophenol + luminol cooxidation. PolyAB activated the cooxidation of p-iodophenol + AAP more effectively and only slightly activated (or inhibited) that of p-iodophenol + luminol. PolyAB diminished the values of rate constants for the interaction of the peroxidase active intermediates, E1 and E2, with p-iodophenol, AAP or luminol. Possible modes of monoAB and polyAB effects on the two substrate pair cooxidation are discussed.

[Optimization of the use of horseradish peroxidase and its antibodies in immunoenzyme analysis]. / Optimizatsiia ispol'zovaniia peroksidazy khrena i ee antitel v immunofermentnom analize.

The steady-state kinetics of peroxidation of 8 aromatic amines was studied. p-Phenylenediamine, o-dianisidine (o-DA) and 3,5,3',5'-tetramethylbenzidine were found to be optimal substrates of horse-radish peroxidase. The kinetics of oxidation of these substrates by horseradish peroxidase modified with three molecules of Strophanthin K was studied as well. Within the temperature range from 37 to 53 degrees C the inactivation rate constants were determined for peroxidase and its conjugate with Strophanthin K. The effect of sugars and polyols on thermal stability of the conjugate peroxidase-Strophanthin K was investigated. A comparative kinetic study was performed of oxidation of o-DA and its conjugate with dextran. The results obtained made a basis for an enzyme immunoassay of cardiac glycosides during their isolation from plant raw material.

[Characteristics of antigen-antibody interaction in the reversed micelles of surface-active agents in heptane]. / Osobennosti vzaimodeistviia "antigen-antitelo" v obrashchennykh mitsellakh poverkhnostno-aktivnykh veshchestv v geptane.

The alterations in the catalytic activity of the horseradish peroxidase after its interaction with antibodies against this enzyme have been studied in buffered solution and in reversed Aerosol OT (AOT) micelles in heptane. The antibodies were obtained by immunizing the rabbits with electrophoretically homogeneous enzyme and were purified by affinity chromatography. In the AOT micelles and mixed micelles containing AOT and Triton X-45, the enzyme interacted with antibodies very rapidly (in less than 5 min), i.e. the micelles did not hinder effective interaction between the enzyme and antibodies. The decrease in the peroxidase catalytic activity upon its interaction with antibodies in a micellar medium was determined by [H2O]/[AOT] ratio, pH and molarity of polar nucleus, as well as by the initial concentration of antibody. In buffered solutions, the decrease n the peroxidase activity of the enzyme--antibody complex was only weakly dependent on pH and molarity of a buffer solution.