Protonation of kanamycin A: Detailing of thermodynamics and protonation sites assignment

Protonation of an aminoglycoside antibiotic kanamycin A sulfate was studied by potentiometric titrations at variable ionic strength, sulfate concentration and temperature. From these results the association constants of differently protonated forms of kanamycin A with sulfate and enthalpy changes for protonation of each amino group were determined. The protonation of all amino groups of kanamycin A is exothermic, but the protonation enthalpy does not correlate with basicity as in a case of simple polyamines. The sites of stepwise protonation of kanamycin A have been assigned by analysis of ¹H-¹³C-HSQC spectra at variable pH in D₂O. Plots of chemical shifts for each H and C atom of kanamycin A vs. pH were fitted to the theoretical equation relating them to pK_a values of ionogenic groups and it was observed that changes in chemical shifts of all atoms in ring C were controlled by ionization of a single amino group with pK_a 7.98, in ring B by ionization of two amino groups with pK_a 6.61 and 8.54, but in ring A all atoms felt ionization of one group with pK_a 9.19 and some atoms felt ionization of a second group with pK_a 6.51, which therefore should belong to amino group at C3 in ring B positioned closer to the ring A while higher pK_a 8.54 can be assigned to the group at C1. This resolves the previously existed uncertainty in assignment of protonation sites in rings B and C.     

Comparative studies on glucose phosphorylating isoenzymes of vertebrates. Identification and characterization of amphibian liver hexokinases

Glucose phosphorylating activities were measured in liver extracts from two urodeles and twenty-six anurans. Fractionation on diethylaminoethyl-cellulose columns of liver extracts from these amphibians permitted the recognition of four hexokinases which are called A, B, C, and D. However, any given amphibian displays only three liver hexokinases and the profiles so far observed are either of the type A-B-D or C-B-D. The distribution of the amphibians in either type of pattern does not show any simple taxonomic relationship. A wide generic and specific, but not individual, variation of the relative proportion of each isoenzyme was observed. Hexokinases A and B were shown to be low K_{m glucose} isoenzymes (0.06 and 0.15 mm glucose, respectively) with normal hyperbolic kinetics. Hexokinase C, also a low K_m isoenzyme (0.05 mm) was found to be inhibited by excess substrate at physiological levels of glucose. Hexokinases A, B, and C were able to phosphorylate fructose, mannose, and 2-deoxyglucose at equal or higher rates than glucose when assayed at saturating sugar levels. Hexokinase D was found to be a high K_m isoenzyme ($\text{K}{}_{0.5}\text{? 2}\text{mM}$) with sigmoidal saturation curves for glucose (Hill coefficient ? 1.6). Fructose and mannose were also phosphorylated by this isoenzyme at about 70% of the glucose rate when studied at saturating sugar concentrations. The properties of the amphibian hexokinases are thus similar, although not identical, to those of mammalian hexokinases.     

Babesia bovis: Proteins of virulent and avirulent parasites passaged through ticks and splenectomized or intact calves

Passage of the avirulent vaccine (K) strain of Babesia bovis (K_A) through either Boophilus microplus ticks, intact calves, or intact calves and then ticks, resulted in two distinct protein and protein antigen profiles as analyzed by two-dimensional gel electrophoresis of biosynthetically labeled proteins and immunoprecipitates. Different degrees of expression of two major acidic antigens of K_A designated Ka₁ (M_r 47,500) and Ka₂ (M_r 43,000) were observed. Ka₁ was apparently lost following passage of K_AB. bovis through intact calves but was strongly represented in the parasite population following a single tick passage. In contrast, passage through ticks of the virulent K_VB. bovis (from which K_A was derived by passage in splenectomized calves) did not lead to strong representation of the Ka₁ protein although there was increased representation of another major acidic protein antigen, designated Kv (M_r 35,000). These data suggest that the previously recognized reversion to a strain-dependent basal antigenic type in the tick vector depends also on intrastrain characteristics such as virulence and strain heterogeneity. The data suggest that K_A is a more heterogeneous population than K_V although cloned isolates are required to establish this point. Comparable syringe passage of another strain of B. bovis, designated C strain, through splenectomized calves resulted in less marked differences between the putative C_A and C_VB. bovis. This may explain the less stable avirulence of C_A compared to K_AB. bovis. Various selection pressures must act, in either the tick or the vertebrate host, on subpopulations in heterogeneous isolates to produce the changes described in protein antigen profiles of B. bovis. The possible relevance of changes in representation of proteins to biological characteristics of B. bovis (such as virulence and tick transmissibility) is discussed.     

The nature of anion inhibition of human red cell carbonic anhydrases.

We studied anionic inhibition of the reaction CO₂ + OH^?? HCO₃^? catalyzed by human red cell carbonic anhydrase B (I) and C (II), using iodide and cyanate. In the forward reaction with respect to CO₂ as the substrate, inhibition was mixed but favoring noncompetitive; the back reaction, with HCO₃^? as the substrate, yielded strict competitive kinetics. Mean inhibition constants, K_I, in the pH range 7.2–7.5 are: iodide, 0.5 mm for enzyme B and 16 mm for C; cyanate, 0.8 μm for B and 20 μm for C. When OH^? was considered as the substrate for the forward reaction, cyanate and chloride behaved as competitive inhibitors. The true inhibition constant (K_I⁰) for cyanate (calculated for infinitely low OH^?) is 0.4 μm for enzyme B and 4 μm for C. Apart from the difference in anion affinity and some 10-fold higher activity of C > B, the isozymes showed similar patterns of inhibition. Data agree with generally proposed mechanisms describing the active site as ZnH₂O with pK_a of about 7.     

Bioactive monoterpene glycosides conjugated with gallic acid from the leaves of Eucalyptus globulus

Two monoterpene glycosides, conjugated with gallic acid [globulusin A (1) and B (2)], together with four known compounds, cypellocarpin A (3), eucaglobulin (4), cuniloside (5) and (1S, 2S, 4R)-trans-2-hydroxy-1,8-cineole beta-d-glucopyranoside (6), were isolated from hot-water extracts of the leaves of Eucalyptus globulus. The structures of compounds 1 and 2 were determined by 1D, 2D NMR and MS spectroscopic analyses. The absolute stereochemistry of 1 was determined by correlating the spectroscopic data with those of synthetic compound 6 with a known configuration. Globulusin A (1) and B (2), cypellocarpin A (3) and eucaglobulin (4), scavenged DPPH free radicals and globulusin A (1) showed a higher antioxidant activity than the other tested compounds, with an IC50 of 3.8microM. Globulusin A (1) and eucaglobulin (4) concentration-dependently suppressed inflammatory cytokine production, tumor-necrosis factor-alpha and interleukin-1beta in cultured human myeloma THP-1 cells co-stimulated with phorbol myristate acetate. These compounds also inhibited melanogenesis in cultured murine melanoma B16F1 cells, without any significant cytotoxicity. These results suggested that globulusin A (1) and eucaglobulin (4), which were isolated as antioxidants from E. globulus, also had anti-inflammatory as well as anti-melanogenesis activity.     

The KC Channel in the cbb3-Type Respiratory Oxygen Reductase from Rhodobacter capsulatus Is Required for Both Chemical and Pumped Protons

The heme-copper superfamily of proton-pumping respiratory oxygen reductases are classified into three families (A, B, and C families) based on structural and phylogenetic analyses. Most studies have focused on the A family, which includes the eukaryotic mitochondrial cytochrome c oxidase as well as many bacterial homologues. Members of the C family, also called the cbb₃-type oxygen reductases, are found only in prokaryotes and are of particular interest because of their presence in a number of human pathogens. All of the heme-copper oxygen reductases require proton-conducting channels to convey chemical protons to the active site for water formation and to convey pumped protons across the membrane. Previous work indicated that there is only one proton-conducting input channel (the K^C channel) present in the cbb₃-type oxygen reductases, which, if correct, must be utilized by both chemical protons and pumped protons. In this work, the effects of mutations in the K^C channel of the cbb₃-type oxygen reductase from Rhodobacter capsulatus were investigated by expressing the mutants in a strain lacking other respiratory oxygen reductases. Proton pumping was evaluated by using intact cells, and catalytic oxygen reductase activity was measured in isolated membranes. Two mutations, N346M and Y374F, severely reduced catalytic activity, presumably by blocking the chemical protons required at the active site. One mutation, T272A, resulted in a substantially lower proton-pumping stoichiometry but did not inhibit oxygen reductase activity. These are the first experimental data in support of the postulate that pumped protons are taken up from the bacterial cytoplasm through the K^C channel.     

Characterization of prolactin-releasing peptide: binding, signaling and hormone secretion in rodent pituitary cell lines endogenously expressing its receptor

The recently discovered prolactin-releasing peptide (PrRP) binds to the PrRP receptor and is involved in endocrine regulation and energy metabolism. However, its main physiological role is currently unknown. Two biologically active isoforms of PrRP exist: the 31 (PrRP31) and the 20 (PrRP20) amino acid forms, which both contain a C-terminal Phe amide sequence. In the present study, the PrRP receptor was immunodetected in three rodent tumor pituitary cell lines: GH3, AtT20 and RC-4B/C cells. The saturation binding of radioiodinated PrRP31 to intact cells demonstrated a K_d in the 10⁻⁹ M range and a B_max in the range of tens of thousands binding sites per cell. For binding to RC-4B/C cells, both PrRP31 and PrRP20 competed with ¹²⁵I-PrRP31 with a similar K_i. The C-terminal analog PrRP13 showed lower binding potency compared to PrRP31 and PrRP20. All PrRP analogs increased the phosphorylation of MAPK/ERK1/2 (mitogen-activated phosphorylase/extracellular-regulated kinase) and CREB (cAMP response element-binding protein) in RC-4B/C cells. Additionally, prolactin release was induced by the PrRP analogs in a dose-dependent manner in RC-4B/C cells. Finally, food intake after intracerebroventricular administration of PrRP analogs in fasted mice was followed. Both PrRP31 and PrRP20 decreased food intake, but PrRP13 did not show significant effect. Studies on pituitary cell lines expressing the PrRP receptor are more physiologically relevant than those on cells transfected with the receptor. This cell type can be used as a model system for pharmacological studies searching for PrRP antagonists and stable effective PrRP agonists, as these drugs may have potential as anti-obesity agents.     

The structural characterization of proteoglycans of culture aortic smooth muscle cells and arterial wall of the pig

Aortic proteoglycans, from the growth medium of cultured smooth muscle cells and from sequential associative and dissociative extracts of the tissue of origin, the pig aorta, were isolated and purified by precipitation with cetylpiridinium chloride. After isopycnic CsCl gradient centrifugation under associative conditions 94% of the cell-secreted proteoglycans were recuperated in the bottom one fifth (?_av = 1.62 g/ml) fraction. In contrast 80% of the tissue proteoglycans of both extracts, fractionated into two fractions: the bottom one fifth (?_av = 1.60 g/ml) fraction and three fifths (?_av = 1.42 g/ml) fraction. Fractionated tissue proteoglycans were composed predominantly of chondroitin sulfate-dermatan sulfate (83–90%) with lower proportions of heparan sulfate (5–11%) and hyaluronic acid (3–6%) whilst cell-secreted proteoglycans showed a similar glycosaminoglycan composition but with a higher proportion of hyaluronic acid (11–13%). Sepharose 2B and C1-2B chromatography of tissue proteoglycans of high buoyant density showed the presence of only subunit proteoglycans whilst those of intermediate density contained a complex species, partially dissociable in 4 M guanidinium chloride, along with K_av 0.50 subunit species. The latter was also observed for cell-secreted proteoglycans although obtained at high buoyant density. The cell-secreted subunit proteoglycans became separated into two distinct populations when chromatographed on Sepharose 4B and C1-4B, half of which eluted in the column V_o and the rest at a K_av of 0.34.. The majority of subunit macromolecules eluted at the V_o fractions of Sepharose 6B and C1-6B columns. Unlike the major species of cartilage proteoglycans, only approx. 20% of purified arterial proteoglycans formed complexes. This proportion could be increased by only 4–7% by interaction, of a mixture of subunit cell-secreted and tissue-extracted proteoglycans, with hyaluronic acid. These results suggest that proteoglycans secreted by cultured aortic smooth muscle cells and present in the aortic tissue possess certain similar physicochemical properties and are present in the form of complex and several subunit species.     

Activation parameters for directly determined first order rate constants for outer sphere electron transfer reactions,and crystal structure of a pertinent pentaammine of CoIII

The outer sphere reductions of Co(NH₃)₅B³⁺ by Fe(CN)₅A³⁻ have been studied. The observed pseudo first order rate constants (Co complex in excess) obey the dependence k_obs=K_osk_et[Co]/(1 +K_os[Co]), as expected for outer sphere electron transfer reactions. Values of the fundamental electron transfer rate constants k_et have been determined, along with the equilibrium constant K_os for a range of reactions in which A and B are pyridyl ligands of different sizes. The first order electron transfer rate constants vary in a manner that is consistcnt with adiabatic electron transfer. The outer sphere ion pairing equilibrium constants K_os have been calculated: K_os=8.6 ± 0.1 × 10² M⁻¹ when A and B=pyridine; K_os=1.07 ± 0.09 × 10³ M⁻¹ where A=pyridine, B=1-phenyl-3-(4-pyridyl)propane; K_os=1.86 ± 0.11 × 10³ M⁻¹ when A=4,4′-bipyridine, B=pyridine; K_os=1.27 ± 0.08 × 10³ M⁻¹ when A=4,4′-bipyridine, B=4-phenylpyridine. Distances of closest approach between the metal centers in the reactive ion pairs are compared, and it is concluded that there is a common mechanism, in which the ammonia side of the cobalt complex approaches the cyano side of the iron complex in each reactive ion pair.The distance of closest approach between the two metal centers (a) was calculated from the experimental values for the ion pairing equilibrium constant K_os at 25 °C: 5.2 Å when A=4,4′-bipyridine, B=pyridine; 5.4 Å when A=4,4′-bipyridine, B=4-phenylpyridine; 5.5 Å when A=pyridine, B=1-phenyl-3-(4-pyridyl)propane; 5.7 Å when A=B=pyridine. These relatively short metal-metal distances, when compared to the X-ray structure of the compound [Co(NH₃)₅(4-phenylpyridine)]₂[S₂O₆]₃· 4H₂O, do not support an ion pair orientation in which the two substituted pyridine ligands A and B are oriented toward each other. [P2₁/c,a=7.399(3), b=22.355(10), c=13.776(4) Å, β=92.02(3)°, R=0.070.] The crystallographic results show that if the two pseudo-octahedral coordination spheres are oriented in the reactive ion pair so that an ammonia face of the cobalt complex is at hydrogen bonding distance from a cyano face on the iron complex, the metal-metal distance is 5.3 Å, a distance which is in agreement with the kinetic results.     

Ionic interactions for substituted MCH1R inhibitors studied by pKa values

MCH1R inhibitors with the quinoline moiety having the aromatic amine and aliphatic amine chain were selected, and then the effect of substituents of the quinoline ring on the ionic interaction were studied by calculating pK_a values for these amines at the B3LYP/6-311++G(d,p)//B3LYP/6-31+G(d) level in the gas phase and in water. For substituent with C, N, O, and S atoms next to the quinoline ring, respectively, the pK_a values of aromatic amines are estimated to be 8.98, 12.19, 4.64, and 4.33 and those of the aliphatic amines are 12.65, 10.82, 9.94, and 11.55, respectively.     

The Combination of Vitamin K3 and Vitamin C Has Synergic Activity against Forms of Trypanosoma cruzi through a Redox Imbalance Process

Chagas’ disease is an infection that is caused by the protozoan Trypanosoma cruzi, affecting millions of people worldwide. Because of severe side effects and variable efficacy, the current treatments for Chagas’ disease are unsatisfactory, making the search for new chemotherapeutic agents essential. Previous studies have reported various biological activities of naphthoquinones, such as the trypanocidal and antitumor activity of vitamin K₃. The combination of this vitamin with vitamin C exerted better effects against various cancer cells than when used alone. These effects have been attributed to an increase in reactive oxygen species generation. In the present study, we evaluated the activity of vitamin K₃ and vitamin C, alone and in combination, against T. cruzi. The vitamin K₃ + vitamin C combination exerted synergistic effects against three forms of T. cruzi, leading to morphological, ultrastructural, and functional changes by producing reactive species, decreasing reduced thiol groups, altering the cell cycle, causing lipid peroxidation, and forming autophagic vacuoles. Our hypothesis is that the vitamin K₃ + vitamin C combination induces oxidative imbalance in T. cruzi, probably started by a redox cycling process that leads to parasite cell death.     

Identification and characterization of a 20β-HSDH from the anaerobic gut bacterium Butyricicoccus desmolans ATCC 43058

Members of the gastrointestinal microbiota are known to convert glucocorticoids to androstanes, which are subsequently converted to potent androgens by other members of the gut microbiota or host tissues. Butyricicoccus desmolans and Clostridium cadaveris have previously been reported for steroid-17,20-desmolase and 20β-hydroxysteroid dehydrogenase (HSDH) activities that are responsible for androstane formation from cortisol; however, the genes encoding these enzymes have yet to be reported. In this work, we identified and located a gene encoding 20β-HSDH in both B. desmolans and C. cadaveris. The 20β-HSDH of B. desmolans was heterologously overexpressed and purified from Escherichia coli. The enzyme was determined to be a homotetramer with subunit molecular mass of 33.8 ± 3.7 kDa. The r20β-HSDH displayed pH optimum in the reductive direction at pH 9.0 and in the oxidative direction at pH 7.0–7.5 with (20β-dihydro)cortisol and NAD(H) as substrates. Cortisol is the preferred substrate with K_m, 0.80 ± 0.06 μM; V_max, 30.36 ± 1.97 μmol·min⁻¹; K_cat, 607 ± 39 μmol·μM⁻¹·min⁻¹; K_cat/K_m, 760 ± 7.67. Phylogenetic analysis of the 20β-HSDH from B. desmolans suggested that the 20β-HSDH is found in several Bifidobacterium spp., one of which was shown to express 20β-HSDH activity. Notably, we also identified a novel steroid-17,20-desmolase-elaborating bacterium, Propionimicrobium lymphophilum, a normal inhabitant of the urinary tract.     

Rates of Transformation of Methyl Parathion and Diethyl Phthalate by Aufwuchs Microorganisms

Using batch cultures, we determined transformation rates for low concentrations of two toxicants—an insecticide, methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate), and a plasticizer, diethyl phthalate—by aufwuchs, aquatic microbial growth attached to submerged surfaces or suspended in streamers or mats. Aufwuchs samples were collected from field sites, an indoor channel, and a continuous-flow fermentor. Aufwuchs fungi, protozoa, and algae did not transform methyl parathion or diethyl phthalate, but bacteria rapidly transformed both chemicals. Second-order transformation rate coefficients, K_b, based on total plate counts of bacteria in aufwuchs, were determined for potential use in a mathematical model capable of predicting the transport and fate of chemicals in aquatic systems. K_b for both methyl parathion and diethyl phthalate decreased as the concentration of total bacteria, [B], increased in aufwuchs. This effect resulted from the proportion of nontransformer to transformer bacteria increasing as [B] increased and from the rate of transformation per transformer cell decreasing as [B] increased. First-order transformation rate coefficients, K₁, were relatively stable per unit of surface area colonized by aufwuchs, because K_b decreased as [B] increased (K₁ = K_b × [B]).     

Potassium-Dependent Increase in RNA and Protein Synthesis in the Early Phase of Incubation of the Thermodormant Phacelia tanacetifolia Seeds

The seeds of Phacelia tanacetifolia Benth. cv Bleu Clair incubated at 30°C in the dark did not germinate and did not activate K⁺ uptake capacity. The administration of 1 millimolar K⁺ in the early phase of incubation stimulated RNA and protein synthesis. The possible role of K⁺ in promoting the marcromolecular syntheses during the early phase of germination is discussed.     

Demonstration of ΔpH- and Δψ-induced synthesis of inorganic pyrophosphate in chromatophores from Rhodospirillum rubrum

It is possible to obtain synthesis of PP_i by artifical ion potentials in Rhodospirillum rubrum chromatophores. PP_i can be formed by K⁺-diffusion gradients (Δψ), H⁺ gradients (ΔpH) or a combination of both. In contrast, ATP can only be synthesized by imposed Δψ or Δψ+ΔpH. For ATP formation there is also a threshold value of K⁺ concentration below which synthesis of ATP is not possible. Such a threshold is not found for PP_i formation. Both PP_i and ATP syntheses are abolished by addition of FCCP or nigericin and only marginally affected by electron transport inhibitors. The synthesis of PP_i can be monitored for several minutes before it ceases, while ATP production stops within 30 s. As a result the maximal yield of PP_i is 200 nmol PP_i/μmol BChl, while that of ATP is no more than 25 nmol ATP/μmol BChl. The initial rates of syntheses were 0.50 μmol PP_i/μmol BChl per min and 2.0 μmol ATP/μmol per min, respectively. These rates are approx. 50 and 20% of the respective photophosphorylation rates under saturating illumination.     

Characterization of the enzyme responsible for nopaline and ornaline synthesis in sunflower crown gall tissues

Extracts prepared from sunflower (Helianthus annuus L.) crown gall tissues induced by Agrobacterium tumefaciens strains C58 and T37 (nopaline utilizers) catalyze the synthesis of nopaline and ornaline. These compounds are not synthesized in extracts of crown gall tissues induced by strains B6, 15955 (octopine utilizers), and AT1 (utilizes neither octopine nor nopaline) or in extracts of habituated sunflower callus. Both synthetic activities require NADPH, α-ketoglutarate, and either arginine or ornithine; histidine and lysine will not substitute. Incorporation of arginine or ornithine into product is inhibited by the other substrate but not by histidine or lysine. On the basis of inhibition and K_m data, both activities appear to be catalyzed by one enzyme and the same enzyme is apparently present in crown gall tissues induced by strains C58 and T37.     

New Insights on the Mechanism of the K+-Independent Activity of Crenarchaeota Pyruvate Kinases

Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K⁺-dependent, whereas lysine confers K⁺-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K⁺-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K⁺ or an internal positive charge, the Thermofilum pendens pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K⁺-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K⁺ or the mutant E117K in the absence of K⁺. Thus, the substrate binding order of the T. pendens enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (T_m of 99.2°C), and the other (T_m of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (T_m of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in Pyrobaculum aerophilum and modeled in T. pendens were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the T. pendens pyruvate kinase could allow charge-independent catalysis.     

Spectroscopic study of the complexation of benzo-15-crown-5 and dibenzo-30-crown-10 with sodium and potassium ions in binary acetonitrile-water mixture

The interaction between Na⁺ and K⁺ ions and benzo-15-crown-5 and dibenzo-30-crown-10 in CH_3⁻ CNH₂O mixtures has been studied spectrophotometrically, at 25 °C and at ionic strength of 0.015. The formation constants for 1:1 complexes at different weight percentages of acetonitrile in water were determined. In all solvent mixtures used, the stabilities of the complexes vary in the order DB30C10-K⁺ > B15C5-K⁺ > B15C5-Na⁺. A linear relationship is observed between log K_f of the three complexes and the mole fraction of acetonitrile. The solvent dependence of the stability constants is most notable with B15C5-K⁺ and the least with DB30C10-K⁺ complex.     

Analysis of phospholipase C (Bacillus cereus) action toward mixed micelles of phospholipid and surfactant.

The action of phospholipase C (Bacillus cereus) toward mixed micelles of phosphatidylcholine and the nonionic surfactant Triton X-100 is analyzed according to the “surfaceas-cofactor” kinetic scheme recently proposed for characterizing the action of lipolytic enzymes [Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem.250, 9013–9020]. According to this scheme, the enzyme first associates with the surface or mixed micelles, where the dissociation constant is K_s^A. The enzyme, now part of the mixed micelle surface, then binds the substrate phospholipid molecule in its active site and this binding is related to the Michaelis constant, K_m^B. The surface, or mixed micelles in this scheme, behaves kinetically as a cofactor in that, under initial rate conditions, the surface properties of the mixed micelles are virtually unchanged after catalysis. For phospholipase C with egg phosphatidylcholine as substrate, it was found that at pH 6.4 (the pH optimum for the enzyme) and 40 °C, V is about 2 × 10³ μmol min^?1 (mg of protein)^?1. K_s^A is about 2 mm and K_m^B is 1 to 2 × 10^?10 mol cm^?2. The kinetic constants for phospholipase C are compared with those previously reported for phospholipase A₂ and the membrane-bound enzyme phosphatidylserine decarboxylase determined under similar conditions.     

Electrostatic analysis of the hepatitis C virus NS3 helicase reveals both active and allosteric site locations

Multi-conformation continuum electrostatics (MCCE) was used to analyze various structures of the NS3 RNA helicase from the hepatitis C virus in order to determine the ionization state of amino acid side chains and their pK_as. In MCCE analyses of HCV helicase structures that lacked ligands, several active site residues were identified to have perturbed pK_as in both the nucleic acid binding site and in the distant ATP-binding site, which regulates helicase movement. In all HCV helicase structures, Glu493 was unusually basic and His369 was abnormally acidic. Both these residues are part of the HCV helicase nucleic acid binding site, and their roles were analyzed by examining the pH profiles of site-directed mutants. Data support the accuracy of MCCE predicted pK_a values, and reveal that Glu493 is critical for low pH enzyme activation. Several key residues, which were previously shown to be involved in helicase-catalyzed ATP hydrolysis, were also identified to have perturbed pK_as including Lys210 in the Walker-A motif and the DExD/H-box motif residues Asp290 and His293. When DNA was present in the structure, the calculated pK_as shifted for both Lys210 and Asp290, demonstrating how DNA binding might lead to electrostatic changes that stimulate ATP hydrolysis.