Conversion of 5-S-methyl-5-thio-D-ribose to methionine in Klebsiella pneumoniae. Stable isotope incorporation studies of the terminal enzymatic reactions in the pathway

Extracts of Klebsiella pneumoniae convert 5-S-methyl-5-thio-D-ribose (methylthioribose) to methionine and formate. To probe the terminal steps of this biotransformation, [1-13C]methylthioribose has been synthesized and its metabolism examined. When supplemented with Mg2+, ATP, L-glutamine, and dioxygen, cell-free extracts of K. pneumoniae converted 50% of the [1-13C]methylthioribose to [13C]formate. The formation of [13C]formate was established by 13C and 1H NMR spectroscopy studies of the purified formate, and by 13C and 1H NMR spectroscopy and mass spectrometry studies of its p-phenylphenacyl derivative. By contrast, no incorporation of label from [1-13C]methylthioribose into the biosynthesized methionine was detected by either mass spectrometry or 13C and 1H NMR spectroscopy. The most reasonable interpretation of these results is that C-1 of methylthioribose is converted directly to formate concomitant with the conversion of carbon atoms 2-5 to methionine. The penultimate step in the conversion of methylthioribose to methionine and formate is an oxidative carbon-carbon bond cleavage reaction in which an equivalent of dioxygen is consumed. To investigate the fate of the dioxygen utilized in this reaction, the metabolism of [1-13C]methylthioribose in the presence of 18O2 was also examined. Mass spectrometry revealed the biosynthesis of substantial amounts of both [18O1]methionine and [13C, 18O1]formate under these conditions. These results suggest that the oxidative transformation in the conversion of methylthioribose to methionine and formate may be catalyzed by a novel intramolecular dioxygenase. A mechanism for this dioxygenase is proposed.     

Formation of reduced nicotinamide adenine dinucleotide peroxide

Incubation of NADH at neutral and slightly alkaline pH leads to the gradual absorption of 1 mol of H+. This uptake of acid requires oxygen and mainly yields anomerized NAD+ (NAD+), with only minimal formation od acid-modified NADH. The overall stoichiometry of the reaction is: NADH + H+ + 1/2O2 leads to H2O + NAD+, with NADH peroxide (HO2-NADH+) serving as the intermediate that anomerizes and breaks down to give NAD+ and H2O2. The final reaction reaction mixture contains less than 0.1% of the generated H2O2, which is nonenzymically reduced by NADH. The latter reaction is inhibited by catalase, leading to a decrease in the overall rate of acid absorption, and stimulated by peroxidase, leading to an increase in the overall rate of acid absorption. Although oxygen can attack NADH at either N-1 or C-5 of the dihydropyridine ring, the attack appears to occur primarily at N-1. This assignment is based on the inability of the C-5 peroxide to anomerize, whereas the N-1 peroxide, being a quaternary pyridinium compound, can anomerize via reversible dissociation of H2O2. The peroxidase-catalyzed oxidation of NADH by H2O2 does not lead to anomerization, indicating that anomerization occurs prior to the release of H2O2. Chromatography of reaction mixtures on Dowex 1 formate shows the presence of two major and several minor neutral and cationic degradation products. One of the major products is nicotinamide, which possibly arises from breakdown of nicotinamide-1-peroxide. The other products have not been identified, but may be derived from other isomeric nicotinamide peroxides.     

Characterization of recombinant Aspergillus fumigatus mannitol-1-phosphate 5-dehydrogenase and its application for the stereoselective synthesis of protio and deuterio forms of D-mannitol 1-phosphate

A putative long-chain mannitol-1-phosphate 5-dehydrogenase from Aspergillus fumigatus (AfM1PDH) was overexpressed in Escherichia coli to a level of about 50% of total intracellular protein. The purified recombinant protein was a approximately 40-kDa monomer in solution and displayed the predicted enzymatic function, catalyzing NAD(H)-dependent interconversion of d-mannitol 1-phosphate and d-fructose 6-phosphate with a specific reductase activity of 170 U/mg at pH 7.1 and 25 degrees C. NADP(H) showed a marginal activity. Hydrogen transfer from formate to d-fructose 6-phosphate, mediated by NAD(H) and catalyzed by a coupled enzyme system of purified Candida boidinii formate dehydrogenase and AfM1PDH, was used for the preparative synthesis of d-mannitol 1-phosphate or, by applying an analogous procedure using deuterio formate, the 5-[2H] derivative thereof. Following the precipitation of d-mannitol 1-phosphate as barium salt, pure product (>95% by HPLC and NMR) was obtained in isolated yields of about 90%, based on 200 mM of d-fructose 6-phosphate employed in the reaction. In situ proton NMR studies of enzymatic oxidation of d-5-[2H]-mannitol 1-phosphate demonstrated that AfM1PDH was stereospecific for transferring the deuterium to NAD+, producing (4S)-[2H]-NADH. Comparison of maximum initial rates for NAD+-dependent oxidation of protio and deuterio forms of D-mannitol 1-phosphate at pH 7.1 and 25 degrees C revealed a primary kinetic isotope effect of 2.9+/-0.2, suggesting that the hydride transfer was strongly rate-determining for the overall enzymatic reaction under these conditions.     

Secondary structure analysis of HIV-1-gp41 in solution and adsorbed to aluminum hydroxide by Fourier transform infrared spectroscopy

The formulation of human vaccines often includes adjuvants such as aluminum hydroxide that are added to enhance the immune responses to vaccine antigens. However, these adjuvants may also affect the conformation of antigenic proteins. Such structural modifications could lead to changes in antigenicity such that suboptimal protective immune responses could be generated relative to those induced by the vaccine antigens alone. Here, we used attenuated total reflectance infrared spectroscopy (ATR-FTIR) to compare the secondary structures of recombinant HIV-1-gp41 (gp41) in solution or adsorbed to aluminum hydroxide. The gp41 secondary structure content was 72% alpha-helices and 28% beta-sheets in 5 mM formate buffer p(2)H 2.5, while it was 66% beta-sheets and 34% random coil in acetonitril/(2)H(2)O (95/5:v/v). A fully reversible conformational change of gp41 in acetonitril/(2)H(2)O (95/5:v/v) was observed upon addition of either 35 mM formate p(2)H 2.5 or 0.1% (w/v) detergent (Tween 20, Hecameg, Brij 35 or beta-d-octyl-glucopyranoside). When gp41 was adsorbed to aluminum hydroxide in the presence of 0.1% (w/v) detergent, in either formate or in acetonitril/(2)H(2)O (95/5:v/v) its secondary structure remained stable and was identical to that of gp41 in 5 mM formate buffer p(2)H 2.5. The method described here could be applied for the characterization of gp41 conformers for use in immunological screening of antigens, and more generally to all antigenic proteins adsorbed to aluminum hydroxide.     

Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays utilizing formate as an alternate substrate for bicarbonate.

Formate is an alternate substrate for bicarbonate in the reaction with PEP catalyzed by phosphoenolpyruvate carboxylase from Zea mays, producing formyl phosphate and pyruvate. The Km for formate is 25 +/- 2 mM, and the maximum velocity is 1% of that for bicarbonate at pH 8.0. Use of [18O]formate produces inorganic phosphate containing 1 equiv of 18O, but no label is incorporated into residual phosphoenolpyruvate. PEP carboxylase catalyzes the hydrolysis of phosphoglycolate or L-phospholactate 2000 times more slowly and D-phospholactate 4000 times more slowly than the reaction between bicarbonate and PEP.     

A Methanocaldococcus jannaschii archaeal signature gene encodes for a 5-formaminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate synthetase. A new enzyme in purine biosynthesis

We have identified and characterized a new member of the ATP-grasp enzyme family that catalyzes the ATP- and formate-dependent formylation of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (AICAR) to 5-formaminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (FAICAR) in the absence of folates. The enzyme, which we designate as PurP, is the product of the Methanocaldococcus jannaschii purP gene (MJ0136), which is a signature gene for Archaea. As is characteristic of reactions catalyzed by this family of enzymes, the other products of the reaction, ADP and P(i), were produced stoichiometrically with the amount of ATP, formate, and AICAR used. Formyl phosphate was found to substitute for ATP and formate in the reaction, yet the methylene analog, phosphonoacetaldehyde, was not an inhibitor or substrate for the reaction. The enzyme, along with PurO, which catalyzes the cyclization of FAICAR to inosine 5'-monophosphate, catalyzes the same overall transformation in purine biosynthesis as is accomplished by PurH in bacteria and eukaryotes. No homology exists between PurH and either PurO or PurP. 1H NMR and gas chromatography-mass spectrometry analysis of an M. jannaschii cell extract showed the presence of free formate that can be used by the enzyme for purine biosynthesis. This formate arises by the reduction of CO2 with hydrogen; this was demonstrated by incorporating 13C into the formate when M. jannaschii cell extracts were incubated with H13CO3- and hydrogen gas. The presence of this signature gene in all of the Archaea indicates the presence of a purine biosynthetic pathway proceeding in the absence of folate coenzymes.     

Variation of transition-state structure as a function of the nucleotide in reactions catalyzed by dehydrogenases. 2. Formate dehydrogenase

Since hydride transfer is completely rate limiting for yeast formate dehydrogenase [Blanchard, J.S., & Cleland, W. W. (1980) Biochemistry 19, 3543], the intrinsic isotope effects on this reaction are fully expressed. Primary deuterium, 13C, and 18O isotope effects in formate and the alpha-secondary deuterium isotope effect at C-4 of the nucleotide have been measured for nucleotide substrates with redox potentials varying from -0.320 (NAD) to -0.258 V (acetylpyridine-NAD). As the redox potential gets more positive, the primary deuterium isotope effect increases from 2.2 to 3.1, the primary 13C isotope effect decreases from 1.042 to 1.036, the alpha-secondary deuterium isotope effect drops from 1.23 to 1.06, and Vmax decreases. The 18O isotope effects increase from 1.005 to 1.008 per single 18O substitution in formate (these values are dominated by the normal isotope effect on the dehydration of formate during binding; pyridinealdehyde-NAD gives an inverse value, possibly because it is not fully dehydrated during binding). These isotope effects suggest a progression toward earlier transition states as the redox potential of the nucleotide becomes more positive, with NAD having a late and acetyl-pyridine-NAD a nearly symmetrical transition state. By contrast, the I2 oxidation of formate in dimethyl sulfoxide has a very early transition state (13k = 1.0154; Dk = 2.2; 18k = 0.9938), which becomes later as the proportion of water in the solvent increases (13k = 1.0265 in 40% dimethyl sulfoxide and 1.0362 in water). alpha-secondary deuterium isotope effects with formate dehydrogenase are decreased halfway to the equilibrium isotope effect when deuterated formate is the substrate, showing that the bending motion of the secondary hydrogen is coupled to hydride transfer in the transition state and that tunneling of the two hydrogens is involved. The 15N isotope effect of 1.07 for NAD labeled at N-1 of the nicotinamide ring suggests that N-1 becomes pyramidal during the reaction. 18O fractionation factors for formate ion relative to aqueous solution are 1.0016 in sodium formate crystal, 1.0042 bound to Dowex-1, and 1.0040 as an ion pair (probably hydrated) in CHCl3. The CO2 analogue azide binds about 10(4) times better than the formate analogue nitrate to enzyme-nucleotide complexes (even though the Ki values for both and the affinity for formate vary by 2 orders of magnitude among the various nucleotides), but the ratio is not sensitive to the redox potential of the nucleotide. Thus, not the nature of the transition state but rather the shape of the initial binding pocket for formate is determining the relative affinity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence from 18O exchange measurements for steps involving a weak acid and a slow chemical transformation in the mechanism of phosphorylation of the gastric H+, K+-ATPase by inorganic phosphate

Phosphorylation of the gastric H,K-ATPase by Pi has been studied by measuring the P18Oj16O4-j distribution as a function of time at different H+, K+, and [18O]Pi concentrations. The advantage of isotope exchange measurements is that the P18Oj16O4-j distribution depends on the relative rates of HOH loss to form the phosphoenzyme intermediate and Pi dissociation from the enzyme. Therefore, 18O exchange is a sensitive probe of mechanism. K+ increases the exchange rate (v(ex] but does not affect the partition coefficient (Pc) that determines the P18Oj16O4-j distribution. Conversely, H+ inhibits exchange. A single Pc describes the data at every pH, but the value increases from 0.04 at pH 8 to 0.64 at pH 5.5. Vex depends hyperbolically on [Pi]0. Km for Pi does not depend on pH, and Pc does not depend on [Pi]0. Individual rate constants in the phosphorylation mechanism are estimated. Formation of the E.Pi complex that looses HOH is 1-2 orders of magnitude slower at pH 5.5 than at pH 8 and is not diffusion controlled. The observed change in Pc with pH is compatible with catalysis occurring by a different mechanism when a group with pKa = 7.2 is protonated. Slower than diffusion-controlled formation of the E.Pi complex that splits out HOH is evidence for a relatively slow, unimolecular chemical transformation involving an additional intermediate in the phosphorylation mechanism, such as a protein conformational change.     

Iron and xanthine oxidase catalyze formation of an oxidant species distinguishable from OH.: comparison with the Haber-Weiss reaction

O2- was produced by gamma irradiation of formate solutions, by the action of xanthine oxidase on hypoxanthine and O2, and by the action of ferredoxin reductase on NADPH and paraquat in the presence of O2. Its reaction with H2O2 and various iron chelates was studied. Oxidation of deoxyribose to thiobarbituric acid-reactive products that was appropriately inhibited by OH. scavengers, or formate oxidation to CO2, was used to detect OH(.). With each source of O2-, and by these criteria, Fe(EDTA) efficiently catalyzed this (Haber-Weiss) reaction, but little catalysis was detectable with iron bound to DTPA, citrate, ADP, ATP, or pyrophosphate, or without chelator in phosphate buffer. O2- produced from xanthine oxidase, but not from the other sources, underwent another iron-dependent reaction with H2O2, to produce an oxidant that did not behave as free OH(.). It was formed in phosphate or bicarbonate buffer, and caused deoxyribose oxidation that was readily inhibited by mannitol or Tris, but not by benzoate, formate, or dimethyl sulfoxide. It did not oxidize formate to CO2. Addition of EDTA changed the pattern of inhibition to that expected for a reaction of OH(.). The other chelators all inhibited deoxyribose oxidation, provided their concentrations were high enough. The results are compatible with iron bound to xanthine oxidase catalyzing production of a strong oxidant (which is not free OH.) from H2O2 and O2- produced by the enzyme.     

Respiratory physiology and energy conservation efficiency of Campylobacter jejuni

A study of the electron transport chain of the human intestinal pathogen Campylobacter jejuni revealed a rich complement of b- and c-type cytochromes. Two c-type cytochromes were partially purified: one, possibly an oxidase, bound carbon monoxide whereas the other, of high potential was unreactive with carbon monoxide. Respiratory activities determined with membrane vesicles were 50- to 100-fold higher with formate and hydrogen than with succinate, lactate, malate, or NADH as substrates. Evidence for three terminal respiratory components was obtained from respiratory kinetic studies employing cyanide, and the following Ki values for cyanide were determined from Dixon plots: ascorbate + reduced N,N,N', N'-tetramethyl-p-phenylenediamine, K1 + 3.5 muM; malate, K1 = 55 muM; and hydrogen, K1 = 4.5 muM. Two oxidases (K1 = 90 muM, 4.5 mM) participated in the oxidation of succinate, lactate, and formate. Except with formate, 37 muM HQNO inhibited respiration by approximately 50%. Carbon monoxide had little inhibitory effect on respiration except under low oxygen tension (less than 10% air saturation). The stoichiometry of respiratory-driven proton translocation (H+/O) determined with whole cells was approximately 2 for all substrates examined except hydrogen (H+/) = 3.7) and formate (H+/O = 2.5). The higher stoichiometries observed with hydrogen and formate are consistent with their respective dehydrogenase being located on the periplasmic face of the cytoplasmic membrane. The results of this study suggest that the oxidation of hydrogen and formate probably serves as the major sources of energy for growth.     

Formation of Hydrogen and Formate by Ruminococcus albus

Radioisotopic growth studies with specifically labeled (14)C-glucose confirmed that Ruminococcus albus, strain 7, ferments glucose mainly by the Embden-Myerhof-Parnas pathway to acetate, ethanol, formate, CO(2), H(2), and an unidentified product. Cell suspensions and extracts converted pyruvate to acetate, H(2), CO(2), and a small amount of ethanol. Formate was not produced from pyruvate and was not degraded to H(2) and CO(2), indicating that formate was not an intermediate in the production of H(2) and CO(2) from pyruvate. Cell extract and (14)C-glucose growth studies showed that the H(2)-producing pyruvate lyase reaction is the major route of H(2) and CO(2) production. An active pyruvate-(14)CO(2) exchange reaction was demonstrable with cell extracts. The (14)C-glucose growth studies indicated that formate, as well as CO(2), arises from the 3 and 4 carbon positions of glucose. A formate-producing pyruvate lyase system was not demonstrable either by pyruvate-(14)C-formate exchange or by net formate formation from pyruvate. Growth studies with unlabeled glucose and labeled (14)CO(2) or (14)C-formate suggest that formate arises from the 3 and 4 carbon positions of glucose by an irreversible reduction of CO(2). The results of the studies on the time course of formate production showed that formate production is a late function of growth, and the rate of production, as well as the total amount produced, increases as the glucose concentration available to the organism increases.     

Gamma-radiolysis of N2O-saturated formate solutions. A chain reaction

In the radiolysis of aqueous formate-containing solutions a chain reaction (i, ii) proceeds in the presence of N2O. CO2-. + N2O + H2O----CO2 + N2 + .OH + OH- (i) .OH + HCO2-.----CO2-. + H2O (ii) The chain length depends on the dose rate and the N2O concentration but not on the formate concentration. Typically, G(CO2) approximately 140 molecules (100 eV)-1 is found, with an equivalent amount of N2, at a dose rate of 3 X 10(-3) Gy s-1. The rate constant for the rate-determining step in this chain reaction has been calculated at k(i) = 1600 dm3 mol-1 s-1. The possible relevance of this chain reaction in radiation biological studies is briefly discussed.     

Source of carbon and hydrogen in methane produced from formate by Methanococcus thermolithotrophicus.

Methanococcus thermolithotrophicus is able to produce methane either from H2-CO2 or from formate. The route of formate entry into the methanogenic pathway was investigated by using 2H2O or [13C]formate and analysis by mass spectrometry. When cells (H2-CO2 or formate grown) were transferred to formate medium in 95% 2H water, the proportion of 2H in methane was 95%. When cells (H2-CO2 or formate grown) were transferred to media containing [13C]formate in the presence of H2-CO2 or He-CO2, the ratio of 13CH4 to 12CH4 increased over time parallel to the ratio of 13CO2 to 12CO2. The cells catalyzed a significant exchange of label between [13C]formate and 13CO2.     

Effects of inhibitory ligands on the aerobic carbon monoxide complex of cytochrome c oxidase.

1. In the presence of both CO and O2, ox heart cytochrome c oxidase forms a 607 nm-peak intermediate distinct from both the cytochrome a2+a3 2+CO and the cytochrome a3+a3 2+CO ('mixed-valence') CO complexes. 2. This aerobic CO compound is stable towards ferricyanide addition, but decomposed on treatment with ferric cytochrome a2 ligands such as formate, cyanide and azide. 3. Addition of formate or cyanves rise to a complex with alpha-peak at 598 nm, not identical with any azide complex of the free enzyme, but possibly a cytochrome a3 2+NO complex produced by oxidative attack of partially reduced O2 on the azide. 4. The results support the idea that although the initial reaction of oxygen is with cytochrome a3 2+, the next step is not an oxidation of the ferrous cytochrome a3, but a transfer of O2 to a neighbouring group, such as Cu+, to give Cu2+O2- or similar complexes. 5. The aerobic CO complex is then identified as a3+a3 2+COCu2+O2-; a similar compound ('Compound C') is formed by photolysis of a3+a3 2+CO (the 'mixed-valence' CO complex) in the presence of oxygen at low temperatures.     

Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate

A chitinase (CHT1) and a chitosanase (CHS1) were purified from the culture supernatant of Pseudomonas sp. TKU015 with shrimp shell wastes as the sole carbon and nitrogen source. The optimized conditions of this new species strain (Gen Bank Accession Number EU103629) for the production of chitinases were found to be when the culture was shaken at 30 degrees C for 3 days in 100 mL of medium (pH 8) containing 0.5% shrimp shell powder (SSP) (w/v), 0.1% K2HPO4, and 0.05% MgSO(4).7H2O. The molecular weights of CHT1 and CHS1 determined by SDS-PAGE were approximately 68 kDa and 30 kDa, respectively. The optimum pH, optimum temperature, pH stability, and the thermal stability of CHT1 and CHS1 were pH 6, 50 degrees C, pH 5-7, <50 degrees C and pH 4, 50 degrees C, pH 3-9, <50 degrees C, respectively. CHT1 was inhibited completely by Mn2+ and Fe2+, and CHS1 was inhibited by Mn2+, Cu2+, and PMSF. CHT1 was only specific to chitin substrates, whereas the relative activity of CHS1 increased when the degree of deacetylation of soluble chitosan increased.     

培养基组分对沙打旺(Astragalus adsurgens Pall)组培根增殖的影响及其培养滤液提取物的化感活性

采用L9 (315 )正交设计,研究了B5培养基营养组分对沙打旺组培根增殖的影响;并采用玻璃皿滤纸培养法,对其培养滤液提取物进行生物测定以验证沙打旺组培根的化感活性.结果显示:培养基的所有营养组分中,Fe2 对沙打旺组培根增殖的影响最大,蔗糖、H2PO 4、 Mg2 、 Mn2 、 Cu2 、 Zn2 、 BO3-3、 Co2 、 I-、C8H12ClNO3 C12H18Cl2N4OS C6H5O2N C6H12O6的影响次之,氮、Ca2 、MoO2-4 和NAA的影响最小.根据不同养分条件下沙打旺组培根干重的极差分析,筛选出适宜沙打旺组培根快速增殖的优化培养基.培养滤液提取物的生物测定结果表明沙打旺组培根培养过程中可能产生化感物质;化感作用强度的差异预示营养胁迫可能影响其化感物质的产生.研究为沙打旺组培根再生与繁殖提供一定依据,并揭示养分条件可能是该植物表达化感作用的影响因素.     

Growth of Wolinella succinogenes with polysulphide as terminal acceptor of phosphorylative electron transport

Polysulphide was formed according to reaction (1), when tetrathionate was (1) $${\text{S}}_4 {\text{O}}_6^{2 - } + {\text{HS}}^ - \to 2{\text{S}}_2 {\text{O}}_3^{2 - } + {\text{S(O)}} + {\text{H}}^ + $$ added to an anaerobic buffer (pH 8.5) containing excess sulphide. S(O) denotes the zero oxidation state sulphur in the polysulphide mixture S _infn ^sup2- . The addition of formate to the polysulphide solution in the presence of Wolinella succinogenes caused the reduction of polysulphide according to reaction (2). The bacteria grew in a medium containing formate and sulphide, (2) $${\text{HCO}}_2^ - + {\text{S(O)}} + {\text{H}}2{\text{O}} \to {\text{HCO}}_3^ - + {\text{HS}}^ - + {\text{H}}^ + $$ when tetrathionate was continuously added. The cell density increased proportional to reaction (3) which represents the sum of reactions (1) and (3) $${\text{HCO}}_2^ - + {\text{S}}_{\text{4}} {\text{O}}_6^{2 - } + {\text{H}}2{\text{O}} \to {\text{HCO}}_3^ - + 2{\text{S}}_{\text{2}} {\text{O}}_3^{2 - } + 2{\text{H}}^ + $$ (2). The cell yield per mol formate was nearly the same as during growth on formate and elemental sulphur, while the velocity of growth was greater. The specific activities of polysulphide reduction by formate measured with bacteria grown with tetrathionate or with elemental sulphur were consistent with the growth parameters. The results suggest that W. succinogenes grow at the expense of formate oxidation by polysulphide and that polysulphide is an intermediate during growth on formate and elemental sulphur.     

Stereochemistry of the hydrolysis of the endo isomer of uridine 2',3'-cyclic phosphorothioate catalyzed by the nonspecific phosphohydrolase from Enterobacter aerogenes.

The nonspecific phosphohydrolase from Enterobacter aerogenes (ATCC 13048) requires divalent metal ions for activity, since zinc present in the isolated enzyme can be removed by extensive dialysis against 8-hydroxyquinoline-5-sulfonate at pH 7.5 to yield an inactive enzyme which can be reactivated by addition of Zn2+, Cd2+, Co2+, Mn2+, or Ni2+; six ions of either zinc or cadmium can be incorporated into the inactive enzyme, and this incorporation of metal ion can be correlated with the regaining of activity (J. A. Gerlt, R. Dhesi, and H. C. Hemmings, unpublished experiments). The cadmium-reactivated phosphohydrolase catalyzes the hydrolysis of the endo isomer of uridine 2',3'-cyclic phosphorothioate (U greater than pS) to yield uridine 3'-monophosphorothioate as the major product. After enzymatic hydrolysis of the cyclic phosphorothioate in 19.8% H218O and chemical recyclization of the 18O-labeled acyclic phosphorothioates to yield a mixture of the endo and exo isomers of U greater than pS, 18O is found primarily in the exo isomer, as judged by examination of the 145.7-MHz phosphorus-31 nuclear magnetic resonance spectrum of the mixture. This observation indicates that the cadmium phosphosphohydrolase catalyzes hydrolysis of endo-U greater than pS with inversion of configuration, implying that the hydrolysis reaction proceeds by an in-line attack of water on the phosphorus.     

Insulin-stimulated intracellular hydrogen peroxide production in rat epididymal fat cells.

Insulin stimulation of hydrogen peroxide production by rat epididymal fat cells was investigated by studying the oxidation of formate to CO2 by endogenous catalase. Under optimal concentrations of formate (0.1 to 1 mM) and glucose (0.275 mM), insulin stimulated formate oxidation 1.5- to 2.0-fold. Inhibitors of catalase activity, including nitrite and azide, inhibited both basal and insulin-stimulated formate oxidation at concentrations that did not interfere with insulin effects on glucose C-1 oxidation or glucose H-3 incorporation into lipids. The addition of exogenous catalase increased formate oxidation only slightly, while exogenous H2O2 (0.5 mM) stimulated formate oxidation by endogenous catalase strongly. These data indicate that the insulin-stimulated H2O2 production was intracellular. Insulin dose-response curves for formate oxidation were identical with those for glucose H-3 incorporation into lipids. The dependence of relative insulin effects on the logarithm of the glucose concentration was bell-shaped for formate oxidation and correlated highly with the coresponding dependences of glucose C-1 oxidation and glucose H-3 incorporation into lipids. This suggests that insulin stimulation of intracellular H2O2 production is linked to glucose metabolism. Since it is known that extracellular H2O2 can mimic insulin in several respects, these observations suggest that H2O2 may act as a "second messenger" for the observed effects of insulin.     

Studies on estrogen biosynthesis using radioactive and stable isotopes

The conversion of androgens into estrogen involves three distinct generic reactions which are catalyzed by a single P450 enzyme (aromatase or P450(aromatase)). The first step in the process is the conversion of 19-methyl into a hydroxymethyl group which requires NADPH + O2, thus representing the well-known hydroxylation process. The next stage, converting the -CH2OH into -CHO, also requires NADPH + O2 and may be rationalized either through a second hydroxylation reaction producing a gem-diol, CH(OH)2 (which dehydrates to the aldehyde), or via another route. The final stage in the process again uses NADPH + O2, culminating in the release of C-19 as formate. Our extensive studies using precursors containing 2H, 3H, and 18O have shown that the carbonyl oxygen of the 19-aldehyde group is the one that was introduced in the first step as the hydroxyl group. The aldehydic oxygen along with another, from O2, used in the third step of the process, is incorporated into the released formate. It was found that at each stage of the process, oxygen atoms were introduced or transferred as "whole numbers." In light of these data, mechanisms in which H2O is used to promote the C-10-C-19 bond cleavage or those in which the conversion of the 19-oxoandrostenedione into estrogen is considered to occur via the sequence -CHO----(-)CH(OH)2----estrogen are eliminated. In addition, our mechanistic analysis makes it unlikely that 1 beta-, 2 beta-, or 10 beta-hydroxysteroids serve as intermediates in estrogen biosynthesis. We consider a free radical mechanism for the hydroxylation process.