Sigmoidal Kinetics Research Articles

Kinetic Modeling of the Interactions between 4-Methylumbelliferone, 1-Naphthol, and Zidovudine Glucuronidation by UDP-Glucuronosyltransferase 2B7 (UGT2B7) Provides Evidence for Multiple Substrate Binding and Effector Sites

Interactions between the UGT2B7-catalyzed glucuronidation of zidovudine (AZT), 4-methylumbelliferone (4MU), and 1-naphthol (1NP) were analyzed using multisite and empirical kinetic models to explore the existence of multiple substrate and effector binding sites within this important drug metabolizing enzyme. 4MU and 1NP glucuronidation by UGT2B7 exhibit sigmoidal kinetics characteristic of homotropic cooperativity (autoactivation), which may be modeled assuming the existence of two equivalent, interacting substrate binding sites. In contrast, UGT2B7-catalyzed AZT glucuronidation follows hyperbolic (Michaelis-Menten) kinetics. Although 4MU and 1NP decreased the binding affinity of AZT, the kinetics of AZT glucuronidation changed from hyperbolic to sigmoidal in the presence of both modifiers. Data were well described by a generic two-substrate binding site model in which there is no interaction between the sites in the absence of 4MU or 1NP, but heterotropic cooperativity results from the binding of modifier. Inhibition of 4MU and 1NP glucuronidation by AZT and interactions between 4MU and 1NP required more complex three-site models, where the modifier acts via a distinct effector site to alter either substrate binding affinity or Vmax without affecting the homotropic cooperativity characteristic of 4MU and 1NP glucuronidation. It is noteworthy that 1NP inhibited 4MU glucuronidation, whereas 4MU activated 1NP glucuronidation. The results are consistent with the existence of two "catalytic" sites for each substrate within the UGT2B7 active site, along with multiple effector sites. The multiplicity of binding and effector sites results in complex kinetic interactions between UGT2B7 substrates, which potentially complicates inhibition screening studies.

Substrate Specificity of the 3-Methylcrotonyl Coenzyme A (CoA) and Geranyl-CoA Carboxylases from Pseudomonas aeruginosa

Biotin-containing 3-methylcrotonyl coenzyme A (MC-CoA) carboxylase (MCCase) and geranyl-CoA (G-CoA) carboxylase (GCCase) from Pseudomonas aeruginosa were expressed as His-tagged recombinant proteins in Escherichia coli. Both native and recombinant MCCase and GCCase showed pH and temperature optima of 8.5 and 37 degrees C. The apparent K(0.5) (affinity constant for non-Michaelis-Menten kinetics behavior) values of MCCase for MC-CoA, ATP, and bicarbonate were 9.8 microM, 13 microM, and 0.8 microM, respectively. MCCase activity showed sigmoidal kinetics for all the substrates and did not carboxylate G-CoA. In contrast, GCCase catalyzed the carboxylation of both G-CoA and MC-CoA. GCCase also showed sigmoidal kinetic behavior for G-CoA and bicarbonate but showed Michaelis-Menten kinetics for MC-CoA and the cosubstrate ATP. The apparent K(0.5) values of GCCase were 8.8 microM and 1.2 microM for G-CoA and bicarbonate, respectively, and the apparent K(m) values of GCCase were 10 microM for ATP and 14 microM for MC-CoA. The catalytic efficiencies of GCCase for G-CoA and MC-CoA were 56 and 22, respectively, indicating that G-CoA is preferred over MC-CoA as a substrate. The enzymatic properties of GCCase suggest that it may substitute for MCCase in leucine catabolism and that both the MCCase and GCCase enzymes play important roles in the leucine and acyclic terpene catabolic pathways.

Activation of d-Tyrosine by Bacillus stearothermophilus Tyrosyl-tRNA Synthetase: 2. COOPERATIVE BINDING OF ATP IS LIMITED TO THE INITIAL TURNOVER OF THE ENZYME

The activation of D-tyrosine by tyrosyl-tRNA synthetase has been investigated using single and multiple turnover kinetic methods. In the presence of saturating concentrations of D-tyrosine, the activation reaction displays sigmoidal kinetics with respect to ATP concentration under single turnover conditions. In contrast, when the kinetics for the activation reaction are monitored using a steady-state (multiple turnover) pyrophosphate exchange assay, Michaelis-Menten kinetics are observed. Previous investigations indicated that activation of l-tyrosine by the K233A variant of Bacillus stearothermophilus tyrosyl-tRNA synthetase displays sigmoidal kinetics similar to those observed for activation of d-tyrosine by the wild-type enzyme. Kinetic analyses indicate that the sigmoidal behavior of the d-tyrosine activation reaction is not enhanced when Lys-233 is replaced by alanine. This supports the hypothesis that the mechanistic basis for the sigmoidal behavior is the same for both d-tyrosine activation by wild-type tyrosyl-tRNA synthetase and activation of l-tyrosine by the K233A variant. The observed sigmoidal behavior presents a paradox, as tyrosyl-tRNA synthetase displays an extreme form of negative cooperativity, known as "half-of-the-sites reactivity," with respect to tyrosine binding and tyrosyl-adenylate formation. We propose that the binding of D-tyrosine weakens the affinity with which ATP binds to the functional subunit in tyrosyl-tRNA synthetase. This allows ATP to bind initially to the nonfunctional subunit, inducing a conformational change in the enzyme that enhances the affinity of the functional subunit for ATP. The observation that sigmoidal kinetics are observed only under single turnover conditions suggests that this conformational change is stable over multiple rounds of catalysis.

Kinetic, Spectroscopic, and X-Ray Crystallographic Evidence for the Cooperative Mechanism of the Hydration of Nitriles Catalyzed by a Tetranuclear Ruthenium-μ-oxo-μ-hydroxo Complex

The tetranuclear ruthenium-oxo-hydroxo-hydride complex {[(PCy3)(CO)RuH]4(μ4-O)(μ3-OH)(μ2-OH)} (1) was found to be a highly cooperative catalyst for the nitrile hydration reaction. The cooperative mechanism of the hydration of benzonitrile was established by Hill inhibition kinetics. The treatment of a nitrile substrate with complex 1 led to the catalytically relevant nitrile-coordinated tetraruthenium complex 3. The X-ray structure of the nitrile-coordinated complex 3 showed a considerably “relaxed” tetrameric core structure compared to that of 1. The hydration of para-substituted benzonitriles p-X-C6H4CN with an electron-withdrawing group (X = Cl, Br, CO2H, CF3) exhibited cooperative kinetics, as indicated by the sigmoidal saturation kinetics, while the hydration of nitriles with an electron-donating group (X = OH, OMe, t-Bu, CH3) obeyed Michaelis–Menten saturation kinetics. The formation of a ruthenium hydride species was observed during the hydration of methacrylonitrile, and its monomeric nature was e...

Purification and Kinetic Characterization of 6-Phosphofructo-1-kinase from the Liver of Gilthead Sea Bream (Sparus Aurata)

6-phosphofructo-1-kinase (PFK) was purified to homogeneity from liver of gilthead sea bream (Sparus aurata) and kinetic properties of the enzyme were determined. The native enzyme had an apparent molecular mass of 510 kDa and was composed of 86 kDa subunits, suggesting homohexameric structure. At pH 7, S. aurata liver PFK (PFKL) showed sigmoidal kinetics for fructose-6-phosphate (fru-6-P) and hyperbolic kinetics for ATP. Fructose-2,6-bisphosphate (fru-2,6-P2) converted saturation curves for fru-6-P to hyperbolic and activated PFKL synergistically with AMP. Fru-2,6-P2 counteracted the inhibition caused by ATP, ADP and citrate. Compared to the S. aurata muscle isozyme, PFKL had lower affinity for fru-6-P, higher cooperativity, hyperbolic kinetics in relation to ATP, increased susceptibility to inhibition by ATP, and was less affected by AMP, ADP and inhibition by 3-phosphoglycerate, phosphoenolpyruvate, 6-phosphogluconate or phosphocreatine. The effect of starvation-refeeding on PFKL expression was studied at the levels of enzyme activity and protein content in the liver of S. aurata. Our findings indicate that short-term recovery of PFKL activity after refeeding previously starved fish, may result from allosteric regulation by fru-2,6-P2, whereas combination of activation by fru-2,6-P2 and increase in protein content may determine the long-term recovery of the enzyme activity.

Basis of Arginine Sensitivity of Microbial N -Acetyl- l -Glutamate Kinases: Mutagenesis and Protein Engineering Study with the Pseudomonas aeruginosa and Escherichia coli Enzymes

N-acetylglutamate kinase (NAGK) catalyzes the second step of arginine biosynthesis. In Pseudomonas aeruginosa, but not in Escherichia coli, this step is rate limiting and feedback and sigmoidally inhibited by arginine. Crystal structures revealed that arginine-insensitive E. coli NAGK (EcNAGK) is homodimeric, whereas arginine-inhibitable NAGKs, including P. aeruginosa NAGK (PaNAGK), are hexamers in which an extra N-terminal kinked helix (N-helix) interlinks three dimers. By introducing single amino acid replacements in PaNAGK, we prove the functionality of the structurally identified arginine site, as arginine site mutations selectively decreased the apparent affinity for arginine. N-helix mutations affecting R24 and E17 increased and decreased, respectively, the apparent affinity of PaNAGK for arginine, as predicted from enzyme structures that revealed the respective formation by these residues of bonds favoring inaccessible and accessible arginine site conformations. N-helix N-terminal deletions spanning > or = 16 residues dissociated PaNAGK to active dimers, those of < or = 20 residues decreased the apparent affinity for arginine, and complete N-helix deletion (26 residues) abolished arginine inhibition. Upon attachment of the PaNAGK N-terminal extension to the EcNAGK N terminus, EcNAGK remained dimeric and arginine insensitive. We concluded that the N-helix and its C-terminal portion after the kink are essential but not sufficient for hexamer formation and arginine inhibition, respectively; that the N-helix modulates NAGK affinity for arginine and mediates signal transmission between arginine sites, thus establishing sigmoidal arginine inhibition kinetics; that the mobile alphaH-beta16 loop of the arginine site is the modulatory signal receiver; and that the hexameric architecture is not essential for arginine inhibition but is functionally essential for physiologically relevant arginine control of NAGK.

Promotion of insulin aggregation by protein disulfide isomerase

We examined the aggregation of insulin as a result of reduction of disulfide bonds catalyzed by protein disulfide isomerase (PDI) using various techniques. We demonstrated the kinetic correlation between PDI-catalyzed insulin reduction and the aggregate formation, the relationship between aggregation and amyloid formation, and the structural information on the secondary structure of the aggregates. The initial rate of PDI-catalyzed reduction of insulin, apparent rate constants of aggregate growth for sigmoidal features, and lag times were obtained by changing the PDI concentration, temperature, and insulin concentration. In situ kinetics were studied using the dyes; thioflavin T (ThT) and Congo red (CR) in addition to turbidimetry with the insulin reduction by PDI. The ThT and CR binding assay revealed sigmoidal kinetics, and the spectrum of binding CR showed a red shift against time, suggesting an orderly formation of insulin aggregates. The secondary structure of the PDI-promoted insulin aggregates showed antiparallel β-sheet conformation by FT-IR measurement.

Novel method for quantitative determination of amyloid fibrils of α-synuclein and amyloid β/A4 protein by using resveratrol

Amyloidosis producing insoluble fibrillar protein aggregates is the common pathological feature of various neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases in which α-synuclein and amyloid β/A4 protein (Aβ) participate to form Lewy bodies and senile plaques, respectively. To develop a novel analytical tool for amyloidosis, resveratrol, the major phenolic constituent of red wine and isolatable from grapevines, was employed to monitor the amyloids of α-synuclein and Aβ. Specific interaction to the amyloids enhanced the intrinsic fluorescence of resveratrol at 395 nm with an advent of new shoulder peak at 440 nm following an excitation at 320 nm. An increase in the resveratrol binding fluorescence was proportional to the quantity of amyloids. Typical sigmoidal kinetics of the amyloidosis of α-synuclein assessed with the thioflavin-T binding fluorescence or the β-sheet content was fully reproduced by the resveratrol binding fluorescence. The resveratrol binding to the amyloids became saturated as the dye concentration increased, whereas the enhanced thioflavin-T binding fluorescence was quenched by the unbound thioflavin-T at the high dye concentration. Because resveratrol does not require any adjustment of the amyloid/dye ratio to obtain optimal amyloid binding fluorescence, and it exerts a higher quantum yield than does thioflavin-T, resveratrol is suggested to be a specific and more reliable fluorescent probe to determine the amyloids quantitatively.

Kinetics of Valproic Acid Glucuronidation: Evidence for in Vivo Autoactivation

Sigmoidal or autoactivation kinetics has been observed in vitro for both cytochrome P450- and UDP-glucuronosyltransferase-catalyzed enzymatic reactions. However, the in vivo relevance of sigmoidal kinetics has never been clearly demonstrated. In the current study we investigate the kinetics of valproic acid glucuronide (VPAG) formation both in vivo in adult sheep and in vitro in sheep liver microsomes (pool of 10). After a 100 mg/kg i.v. bolus dose of valproic acid (VPA) to adult sheep (n = 5), the majority of the dose was recovered in urine as VPAG (approximately 79%). Eadie-Hofstee plots of the VPAG formation rate (calculated from urinary excretion rate data for VPAG) were characteristic of autoactivation kinetics and provided estimates of the apparent maximum velocity of an enzymatic reaction (V(max)(app)), the substrate concentration resulting in 50% of V(max)(app) (S(50)(app)), and Hill coefficient (n) of 2.10 +/- 0.75 micromol/min/kg, 117 +/- 56 microM, and 1.34 +/- 0.14, respectively. Comparable estimates of V(max)(app) (2.63 +/- 0.33 micromol/min/kg), S(50)(app) (118 +/- 53 microM), and n (2.06 +/- 0.47) describing overall VPA elimination from plasma were obtained by fitting VPA unbound plasma concentration-time data to a two-compartment model with elimination described by the Hill equation. Consistent with our in vivo observations, Eadie-Hofstee plots of VPAG formation in sheep liver microsomes were characteristic of autoactivation kinetics. To our knowledge, these data provide the first clear demonstration that autoactivation kinetics observed in vitro in liver preparations can translate to the in vivo situation at least under certain experimental conditions and confirm its relevance.

Kinetics of sunflower oil methanolysis at low temperatures

The kinetics of the sunflower oil methanolysis process was studied at lower temperatures (10–30 °C). The sigmoidal kinetics of the process was explained by the mass transfer controlled region in the initial heterogenous regime, followed by the chemical reaction controlled region in the pseudo-homogenous regime. A simple kinetic model, which did not require complex computation of the kinetic constants, was used for simulation of the TG conversion and the FAME formation in the latter regime: the fast irreversible second-order reaction was followed by the slow reversible second-order reaction close to the completion of the methanolysis reaction. The mass transfer was related to the drop size of the dispersed (methanol) phase, which reduced rapidly with the progress of the methanolysis reaction. This was attributed to the formation of the emulsifying agents stabilizing the emulsion of methanol drops into the oil.

Macromolecular crowding and its influence on possible reaction mechanisms in photosynthetic electron flow

The diffusion of plastoquinol and its binding to the Qo site of the cyt bf complex in the course of photosynthetic electron transport was studied by following the sigmoidal flash-induced re-reduction kinetics of P700 after previous oxidation of the intersystem electron carriers. The data resulting from these experiments were matched with a simulation of electron transport using Monte Carlo techniques. The simulation was able to account for the experimental observations. Two different extreme cases of reaction mechanism at the Qo site were compared: a diffusion limited collisional mechanism and a non-diffusion limited tight binding mechanism. Assuming a tight binding mechanism led to best matches due to the high protein density in thylakoids. The varied parameters resulted in values well within the range of published data. The results emphasise the importance of structural characteristics of thylakoids in models of electron transport.

A synchrotron radiation study of the hydrothermal synthesis of layered double hydroxides from MgO and Al2O3 slurries

The hydrothermal reaction of slurries containing MgO and Cp3 alumina has been investigated in situ using energy dispersive X-ray diffraction (EDXRD). A range of temperatures (100, 150, 180 and 240 °C) were studied. Kinetic data for the formation of the hydrotalcite-like Mg–Al layered double hydroxide (LDH) have been determined. At 100 °C the LDH is the predominant phase that is formed but at higher temperatures the impurity phases brucite and boehmite become more significant. The rate of reaction increases with temperature in agreement with Arrhenius behaviour and the LDH growth curves exhibit sigmoidal kinetics. The rate of formation of the LDH phase was found to be approximately equal to the rate of consumption of MgO, indicating that the mechanism of LDH formation is unlikely to proceed via a long-lasting intermediate phase.

Purification, characterization and cloning of aldehyde dehydrogenase from Rhodococcus erythropolis UPV-1

The enzyme responsible for formaldehyde removal in industrial wastewaters by cells of Rhodococcus erythropolis UPV-1 was identified as a broad-specific aldehyde dehydrogenase (EC 1.2.1.3). The enzyme was purified to electrophoretic homogeneity from ethanol-grown cells with a specific activity of 19.5 U mg-1 protein and an activity recovery of 56%. The enzyme showed an isoelectric point (pI) of 5.3 and was a trimer of 162 kDa consisting of three identical 54-kDa subunits. It was specific for NAD+ and showed hyperbolic kinetics for this coenzyme (Km=90 microM), but sigmoidal kinetics for the aliphatic aldehydes used as substrates. The enzyme affinity for aldehydes increased with their hydrocarbon chain length, ranging from 333 microM for formaldehyde to 85 nM for n-octanal. The corresponding calculated Hill coefficients were in the 1.55-2.77 range. With n-propanal as substrate, the optimum pH and temperature for activity were 9.5-10.0 and 47.5 degrees C, respectively, with an Ea for catalysis of 28.6 kJ mol-1. NAD+ protected the enzyme against thermal inactivation, but aldehydes were ineffective. The activity was severely inhibited by p-hydroxymercuribenzoate, indicating that a thiol was essential for catalysis. The 1,524-bp aldhR gene encoding a 507-amino-acid protein was expressed in cells of Escherichia coli M15 as a hexahistidine-tagged protein.

Amino terminal domains of human UDP-glucuronosyltransferases (UGT) 2B7 and 2B15 associated with substrate selectivity and autoactivation

Despite the important role of UDP-glucuronosyltransferases (UGT) in the metabolism of drugs, environmental chemicals and endogenous compounds, the structural features of these enzymes responsible for substrate binding and selectivity remain poorly understood. Since UGT2B7 and UGT2B15 exhibit distinct, but overlapping, substrate selectivities, UGT2B7–UGT2B15 chimeras were constructed here to identify substrate binding domains. A UGT2B7-15-7 chimera that incorporated amino acids 61–194 of UGT2B15 glucuronidated the UGT2B15 substrates testosterone and phenolphthalein, but not the UGT2B7 substrates zidovudine and 11α-hydroxyprogesterone. Derived apparent Km values for testosterone and phenolphthalein glucuronidation by UGT2B7-15(61–194)-7 were similar in magnitude to those determined for UGT2B15. Moreover, glucuronidation of the non-selective substrate 4-methylumbelliferone (4MU) by UGT2B7-15(61–194)-7 and UGT2B15 followed Michaelis–Menten and weak substrate inhibition kinetics, respectively, whereas 4MU glucuronidation by UGT2B7 exhibited sigmoidal kinetics characteristic of autoactivation. Six UGT2B7-15-7 chimeras that incorporated smaller domains of UGT2B15 were subsequently generated. Of these, UGT2B7-15(61–157)-7, UGT2B7-15(91–157)-7 and UGT2B7-15(61–91)-7 glucuronidated 4MU, but activity towards the other substrates investigated here was not detected. Like UGT2B7, the UGT2B7-15(61–157)-7, UGT2B7-15(91–157)-7 and UGT2B7-15(61–91)-7 chimeras exhibited sigmoidal 4MU glucuronidation kinetics. The sigmoidal 4MU kinetic data were well modelled using both the Hill equation and the expression for a two-site model that assumes the simultaneous binding of two substrate molecules at equivalent sites. It may be concluded that residues 61–194 of UGT2B15 are responsible for substrate binding and for conferring the unique substrate selectivity of UGT2B15, while residues 158–194 of UGT2B7 appear to facilitate the binding of multiple 4MU molecules within the active site.

Inorganic carbon limitations on nitrification: Experimental assessment and modelling

Nitrification is a two-step process that involves two different biomass populations: ammonia oxidising biomass (AOB) and nitrite oxidising biomass (NOB). Both populations are autotrophic (i.e. their carbon source is inorganic). Therefore, a deficit of this substrate should result in a decrease of the process rate. Recent technology advances such as the SHARON ® process have brought new scenarios in biological nitrogen removal where these limitations should be considered. Hence, this work examines the inorganic carbon limitation using respirometric and titrimetric techniques. For this aim, the nitrification rate was measured at different total inorganic carbon (TIC) concentrations. The experimental results obtained show that AOB was limited at TIC concentrations lower than 3 mmol C L −1. At the same time, no carbon source limitation for NOB was observed in spite of the low TIC concentrations attained (lower than 0.1 mmol C L −1). The AOB limitation could be successfully modelled using Monod, Tessier and sigmoidal kinetics. The best fit was obtained with sigmoidal kinetics. However, unexpected biomass activity (oxygen consumption) was observed despite a very low TIC concentration (lower than 0.1 mmol C L −1).

Purification and kinetic properties of 6-phosphofructo-1-kinase from gilthead sea bream muscle

The kinetic properties of 6-phosphofructo-1-kinase (PFK) from skeletal muscle (PFKM) of gilthead sea bream ( Sparus aurata) were studied, after 10,900-fold purification to homogeneity. The native enzyme had an apparent molecular mass of 662 kDa and is composed of 81 kDa subunits, suggesting a homooctameric structure. At physiological pH, S. aurata PFKM exhibited sigmoidal kinetics for the substrates, fructose-6-phosphate (fru-6-P) and ATP. Fructose-2,6-bisphosphate (fru-2,6-P 2) converted the saturation curves for fru-6-P to hyperbolic, activated PFKM synergistically with other positive effectors of the enzyme such as AMP and ADP, and counteracted ATP and citrate inhibition. The fish enzyme showed differences regarding other animal PFKs: it is active as a homooctamer, and fru-2,6-P 2 and pH affected affinity for ATP. By monitoring incorporation of 32P from ATP, we show that fish PFKM is a substrate for the cAMP-dependent protein kinase. The mechanism involved in PFKM activation by phosphorylation contrasts with previous observations in other species: it increased V max and did not affect affinity for fru-6-P. Unlike the mammalian muscle enzyme, our findings support that phosphorylation of PFKM may exert a major role during starvation in fish muscle.

Oxidation behavior of the simulated fuel with dissolved fission products in air at 573–873 K

As a part of the DUPIC fuel development program, oxidation behavior of the simulated spent fuel with dissolved fission products has been studied using a thermogravimetry analyzer in the temperature range of 573–873 K in air in order to establish the oxidation temperature and time of the OREOX process during fabrication of DUPIC fuel. Oxidation behavior of an UO 2 sintered pellet has also been measured for the purpose of comparison. From the XRD study, the simulated spent fuel has been converted to U 3O 8, in the temperature range of 573–873 K. The oxidation curves of the simulated spent fuel are displayed sigmoidal reaction kinetics which follows the nucleation-and growth mechanism of the formation of U 3O 8. The fission products forming the solid solution in UO 2 matrix delayed U 3O 8 forming. Activation energies of the simulated spent fuel are higher than those of UO 2. There are transition points in the rate of the oxidation of the simulated fuel and UO 2 between 673 and 723 K, and the activation energies in the low temperature range of 573–673 K are higher than those in the high temperature range of 723–873 K.

A Collision Theory-Based Derivation of Semiempirical Equations for Modeling Dispersive Kinetics and Their Application to a Mixed-Phase Crystal Decomposition

In recent works, the author has shown the utility of new, semiempirical kinetic model equations for treating dispersive chemical processes ranging from slow (minute/hour time scale) solid-state phase transformations to ultrafast (femtosecond) reactions in the gas phase. These two fundamental models (one for homogeneous/deceleratory sigmoidal conversion kinetics and the other for heterogeneous/acceleratory sigmoidal kinetics; isothermal conditions), based on the assumption of a "Maxwell-Boltzmann-like" distribution of molecular activation energies, provide a novel, quantum-based interpretation of the kinetics. As an extension to previous work, it is shown here that the derivation of these dispersive kinetic equations is supported by classical collision theory (i.e., for gas-phase applications). Furthermore, the successful application of the approach to the kinetic modeling of the solid-state decomposition of a binary system, CO2.C2H2, is demonstrated. Finally, the models derived appear to explain some of the (solid-state) kinetic data collected using isoconversional techniques such as those often reported in the thermal analysis literature.

Characterization of Heme-Regulated eIF2α Kinase: Roles of the N-Terminal Domain in the Oligomeric State, Heme Binding, Catalysis, and Inhibition

Heme-regulated eIF2alpha kinase [heme-regulated inhibitor (HRI)] plays a critical role in the regulation of protein synthesis by heme iron. The kinase active site is located in the C-terminal domain, whereas the N-terminal domain is suggested to regulate catalysis in response to heme binding. Here, we found that the rate of dissociation for Fe(III)-protoporphyrin IX was much higher for full-length HRI (1.5 x 10(-)(3) s(-)(1)) than for myoglobin (8.4 x 10(-)(7) s(-)(1)) or the alpha-subunit of hemoglobin (7.1 x 10(-)(6) s(-)(1)), demonstrating the heme-sensing character of HRI. Because the role of the N-terminal domain in the structure and catalysis of HRI has not been clear, we generated N-terminal truncated mutants of HRI and examined their oligomeric state, heme binding, axial ligands, substrate interactions, and inhibition by heme derivatives. Multiangle light scattering indicated that the full-length enzyme is a hexamer, whereas truncated mutants (truncations of residues 1-127 and 1-145) are mainly trimers. In addition, we found that one molecule of heme is bound to the full-length and truncated mutant proteins. Optical absorption and electron spin resonance spectra suggested that Cys and water/OH(-) are the heme axial ligands in the N-terminal domain-truncated mutant complex. We also found that HRI has a moderate affinity for heme, allowing it to sense the heme concentration in the cell. Study of the kinetics showed that the HRI kinase reaction follows classical Michaelis-Menten kinetics with respect to ATP but sigmoidal kinetics and positive cooperativity between subunits with respect to the protein substrate (eIF2alpha). Removal of the N-terminal domain decreased this cooperativity between subunits and affected the other kinetic parameters including inhibition by Fe(III)-protoporphyrin IX, Fe(II)-protoporphyrin IX, and protoporphyrin IX. Finally, we found that HRI is inhibited by bilirubin at physiological/pathological levels (IC(50) = 20 microM). The roles of the N-terminal domain and the binding of heme in the structural and functional properties of HRI are discussed.

New Prospects for an Old Enzyme: Mammalian Cytochrome c Is Tyrosine-Phosphorylated in Vivo

Mammalian cytochrome c (Cyt c) has two primary functions: transfer of electrons from the bc1 complex to cytochrome c oxidase (COX) as part of the mitochondrial electron transport chain (ETC), and participation in type II apoptosis. Several studies have indicated that components of the ETC can be phosphorylated, and we have recently shown that the Cyt c electron acceptor COX is phosphorylated on Tyr-304 of subunit I in liver upon activation of the cAMP-dependent pathway, leading to strong enzyme inhibition. However, covalent modification of Cyt c through phosphorylation has not yet been reported. We have isolated Cyt c from cow heart under conditions that preserve the physiological in vivo phosphorylation status. Western analysis with an anti-phosphotyrosine antibody indicated tyrosine phosphorylation. The site of phosphorylation was definitively assigned by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS) to Tyr-97, one of the four tyrosine residues present in Cyt c. The phosphorylated tyrosine is part of a motif that contains five residues identical to the tyrosine phosphorylation site in COX subunit I. Spectral analysis revealed that the characteristic 695 nm absorption band is shifted to 687 nm and reversed after treatment with alkaline phosphatase. This band results from the Met-80-heme iron bond, and its shift might indicate changes in the catalytic heme crevice. In vivo phosphorylated Cyt c shows enhanced sigmoidal kinetics with COX, and half-maximal turnover is observed at a Cyt c substrate concentration of 5.5 microM compared to 2.5 microM for alkaline phosphatase-treated Cyt c. Possible consequences of Tyr-97 phosphorylation with respect to cardiolipin binding and of location of Tyr-97 in close proximity to Lys-7, a crucial residue for interaction with Apaf-1 during apoptosis, are discussed.