Covalent immobilization of the multienzyme enniatin synthetase

Summary The multienzyme enniatin synthetase was covalently immobilized to N-hydroxysuccinimide activated agarose. The stability of the immobilized enzyme at 25°C was enhanced compared to the soluble enzyme. Immobilization experiments also indicated that the enniatins are synthesized by a single molecule and thus do not require interactions of several enzyme molecules.     

Increased Kit/SCF receptor induced mitogenicity but abolished cell motility after inhibition of protein kinase C.

The product of the c-kit proto-oncogene, denoted Kit/SCF-R, encodes a tyrosine kinase receptor for stem cell factor (SCF). Kit/SCF-R induces proliferation, differentiation or migration of cells within the hematopoietic, gametogenic and melanogenic lineages at different developmental stages. We report here that protein kinase C (PKC) mediates phosphorylation of Kit/SCF-R on serine residues in response to SCF or PMA in intact cells. The phosphorylation inhibits SCF-induced tyrosine autophosphorylation of Kit/SCF-R. In vitro studies showed that PKC phosphorylated the Kit/SCF-R directly on serine residues and inhibited autophosphorylation of Kit/SCF-R, as well as its kinase activity towards an exogenous substrate. The PKC-induced phosphorylation did not affect Kit/SCF-R ligand binding affinity. Inhibition of PKC led to increased SCF-induced tyrosine autophosphorylation, as well as increased SCF-induced mitogenicity. In contrast, PKC was necessary for SCF-induced motility responses, including actin reorganization and chemotaxis. Our data suggest that PKC is involved in a negative feedback loop which regulates the Kit/SCF-R and that the activity of PKC determines whether the effect of SCF will be preferentially mitogenic or motogenic.     

A theoretical study of the<Emphasis Type="Italic"> cis</Emphasis>-dihydroxylation mechanism in naphthalene 1,2-dioxygenase

The catalytic mechanism of naphthalene 1,2-dioxygenase has been investigated by means of hybrid density functional theory. This Rieske-type enzyme, which contains an active site hosting a mononuclear non-heme iron(II) complex, uses dioxygen and two electrons provided by NADH to carry out the cis-dihydroxylation of naphthalene. Since a (hydro)peroxo-iron(III) moiety has been proposed to be involved in the catalytic cycle, it was probed whether and how this species is capable of cis-dihydroxylation of the aromatic substrate. Different oxidation and protonation states of the Fe–O₂ complex were studied on the basis of the crystal structure of the enzyme with oxygen bound side-on to iron. It was found that feasible reaction pathways require a protonated peroxo ligand, Fe^III–OOH; the deprotonated species, the peroxo-iron(III) complex, was found to be inert toward naphthalene. Among the different chemical patterns which have been explored, the most accessible one involves an epoxide intermediate, which may subsequently evolve toward an arene cation, and finally to the cis-diol. The possibility that an iron(V)-oxo species is formed prior to substrate hydroxylation was also examined, but found to implicate a rather high energy barrier. In contrast, a reasonably low barrier might lead to a high-valent iron-oxo species [i.e. iron(IV)-oxo] if a second external electron is supplied to the mononuclear iron center before dioxygenation.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0537-0     

Catalysis by methyl-coenzyme M reductase: a theoretical study for heterodisulfide product formation

Hybrid density functional theory has been used to investigate the catalytic mechanism of methyl-coenzyme M reductase (MCR), an essential enzyme in methanogenesis. In a previous study of methane formation, a scheme was suggested involving oxidation of Ni(I) in the starting square-planar coordination to the high-spin Ni(II) form in the CoM-S-Ni(II)F(430) octahedral intermediate. The methyl radical, concomitantly released by methyl-coenzyme M (CoM), is rapidly quenched by hydrogen atom transfer from the coenzyme B (CoB) thiol group, yielding methane as the first product of the reaction. The present investigation primarily concerns the second and final step of the reaction: oxidation of CoB and CoM to the CoB-S-S-CoM heterodisulfide product and reduction of nickel back to the Ni(I) square-planar form. The activation energy for the second step is found to be around 10 kcal/mol, implying that the first step of methane formation with an activation energy of 20 kcal/mol should be rate-limiting. An oxygen of the Gln147 residue, occupying the rear axial position in the oxidized Ni(II) state, is shown to stabilize the intermediate by 6 kcal/mol, thereby slightly decreasing the barrier for the preceding rate-limiting transition state. The mechanism suggested is discussed in the context of available experimental data. An analysis of the flexibility of the F(430) cofactor during the reaction cycle is also given.     

Proton pumping in cytochrome c oxidase: Energetic requirements and the role of two proton channels

Cytochrome c oxidase is a superfamily of membrane bound enzymes catalyzing the exergonic reduction of molecular oxygen to water, producing an electrochemical gradient across the membrane. The gradient is formed both by the electrogenic chemistry, taking electrons and protons from opposite sides of the membrane, and by proton pumping across the entire membrane. In the most efficient subfamily, the A-family of oxidases, one proton is pumped in each reduction step, which is surprising considering the fact that two of the reduction steps most likely are only weakly exergonic. Based on a combination of quantum chemical calculations and experimental information, it is here shown that from both a thermodynamic and a kinetic point of view, it should be possible to pump one proton per electron also with such an uneven distribution of the free energy release over the reduction steps, at least up to half the maximum gradient. A previously suggested pumping mechanism is developed further to suggest a reason for the use of two proton transfer channels in the A-family. Since the rate of proton transfer to the binuclear center through the D-channel is redox dependent, it might become too slow for the steps with low exergonicity. Therefore, a second channel, the K-channel, where the rate is redox-independent is needed. A redox-dependent leakage possibility is also suggested, which might be important for efficient energy conservation at a high gradient. A mechanism for the variation in proton pumping stoichiometry over the different subfamilies of cytochrome oxidase is also suggested. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

On the observation of a gem diol intermediate after O–O bond cleavage by extradiol dioxygenases. A hybrid DFT study

Catalytic cycle intermediates of a representative extradiol dioxygenase, homoprotocatechuate 2,3-dioxygenase (HPCD), have recently been characterized in crystallo by Kovaleva and Lipscomb. The structures of the identified species indicate that the process of inserting oxygen into the catechol ring occurs stepwise, and involves an Fe(II)-alkylperoxo intermediate and its O–O cleavage product: a gem diol species. In general, these findings corroborate the results of our previous computational studies; however, the fact that the gem diol species is stable enough to be observed in the crystal form seems to be at odds with the computational mechanistic data, which suggest that this intermediate should very readily and spontaneously convert to the epoxide species. The key question then becomes what is actually observed in the X-ray experiments. Here we report additional computational studies undertaken with the hope of clarifying this issue. The results obtained for active site models hosting both the native and the alternative (4-sulfonylcatechol) substrate indicate that the stability of the gem diol species is substantially increased if an electron and a proton are added. If this occurs somehow, the lifetime of the intermediate should be sufficient to observe it.     

A theoretical study on the binding of O(2), NO and CO to heme proteins

The hybrid density functional B3LYP is used to describe the bonding of the diatomic molecules O(2), NO and CO to ferrous heme. Three different models are used, a five-coordinated porphyrin in benzene, the myoglobin active site including the distal histidine and the binuclear center in cytochrome oxidase. The geometric and electronic structures are well described by the B3LYP functional, while experimental binding energies are more difficult to reproduce. It is found that the Cu(B) center in cytochrome oxidase has a similar effect on the binding of the diatomics as the distal histidine in myoglobin.     

A theoretical study on nitric oxide reductase activity in a ba(3)-type heme-copper oxidase

The mechanism of nitric oxide reduction in a ba(3)-type heme-copper oxidase has been investigated using density functional theory (B3LYP). Four possible mechanisms have been studied and free energy surfaces for the whole catalytic cycle including proton and electron transfers have been constructed by comparison to experimental data. The first nitric oxide coordinates to heme a(3) and is partly reduced having some nitroxyl anion character ((3)NO(-)), and it is thus activated toward the attack by the second N-O. In this reaction step a cyclic hyponitrous acid anhydride intermediate with the two oxygens coordinating to Cu(B) is formed. The cyclic hyponitrous acid anhydride is quite stable in a local minimum with high barriers for both the backward and forward reactions and should thus be observable experimentally. To break the N-O bond and form nitrous oxide, the hyponitrous acid anhydride must be protonated, the latter appearing to be an endergonic process. The endergonicity of the proton transfer makes the barrier of breaking the N-O bond directly after the protonation too high. It is suggested that an electron should enter the catalytic cycle at this stage in order to break the N-O bond and form N(2)O at a feasible rate. The cleavage of the N-O bond is the rate limiting step in the reaction mechanism and it has a barrier of 17.3 kcal/mol, close to the experimental value of 19.5 kcal/mol. The overall exergonicity is fitted to experimental data and is 45.6 kcal/mol.