Certhrax Toxin,an Anthrax-related ADP-ribosyltransferase from Bacillus cereus

We identified Certhrax, the first anthrax-like mART toxin from the pathogenic G9241 strain of Bacillus cereus. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we have shown that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. Like anthrax lethal factor, Certhrax was found to require protective antigen for host cell entry. This two-domain enzyme was shown to be 60-fold more toxic to mammalian cells than anthrax lethal factor. Certhrax localizes to distinct regions within mouse RAW264.7 cells by 10 min postinfection and is extranuclear in its cellular location. Substitution of catalytic residues shows that the mART function is responsible for the toxicity, and it binds NAD⁺ with high affinity (K_D = 52.3 ± 12.2 μm). We report the 2.2 Å Certhrax structure, highlighting its structural similarities and differences with anthrax lethal factor. We also determined the crystal structures of two good inhibitors (P6 (K_D = 1.7 ± 0.2 μm, K_i = 1.8 ± 0.4 μm) and PJ34 (K_D = 5.8 ± 2.6 μm, K_i = 9.6 ± 0.3 μm)) in complex with Certhrax. As with other toxins in this family, the phosphate-nicotinamide loop moves toward the NAD⁺ binding site with bound inhibitor. These results indicate that Certhrax may be important in the pathogenesis of B. cereus.     

Effectors of rat-heart hexokinases and the control of rates of glucose phosphorylation in the perfused rat heart

1. The kinetic properties of the soluble and particulate hexokinases from rat heart have been investigated. 2. For both forms of the enzyme, the K_m for glucose was 45μm and the K_m for ATP 0·5mm. Glucose 6-phosphate was a non-competitive inhibitor with respect to glucose (K_i 0·16mm for the soluble and 0·33mm for the particulate enzyme) and a mixed inhibitor with respect to ATP (K_i 80μm for the soluble and 40μm for the particulate enzyme). ADP and AMP were competitive inhibitors with respect to ATP (K_i for ADP was 0·68mm for the soluble and 0·60mm for the particulate enzyme; K_i for AMP was 0·37mm for the soluble and 0·16mm for the particulate enzyme). P_i reversed glucose 6-phosphate inhibition with both forms at 10mm but not at 2mm, with glucose 6-phosphate concentrations of 0·3mm or less for the soluble and 1mm or less for the particulate enzyme. 3. The total activity of hexokinase in normal hearts and in hearts from alloxan-diabetic rats was 21·5μmoles of glucose phosphorylated/min./g. dry wt. of ventricle at 25°. The temperature coefficient Q₁₀ between 22° and 38·5° was 1·93; the ratio of the soluble to the particulate enzyme was 3:7. 4. The kinetic data have been used to predict rates of glucose phosphorylation in the perfused heart at saturating concentrations of glucose from measured concentrations of ATP, glucose 6-phosphate, ADP and AMP. These have been compared with the rates of glucose phosphorylation measured with precision in a small-volume recirculation perfusion apparatus, which is described. The correlation between predicted and measured rates was highly significant and their ratio was 1·07. 5. These findings are consistent with the control of glucose phosphorylation in the perfused heart by glucose 6-phosphate concentration, subject to certain assumptions that are discussed in detail.     

Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 4·7 μm) and hypoxanthine phosphoribosyltransferase (K_i 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of K_i for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.     

Transport of glucose and galactose in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25° was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S]_i) and extracellular ([S]_o) glucose concentrations was increased by 0·4mm-phlorrhizin and 0·3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S]_i/[S]_o fell below 1·0 only at [S]_o higher than 0·5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K_m 1·16mm; V_max. 4·5μmoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0·1mm-2,4-dinitrophenol, 0·4mm-phlorrhizin and by the absence of external Na⁺. 6. The kinetic parameters of galactose entry into the cells were: K_m 1·5mm; V_max 10μmoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0°, was inhibited by 0·4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na⁺-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na⁺-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

C3larvin Toxin,an ADP-ribosyltransferase from Paenibacillus larvae

C3larvin toxin was identified by a bioinformatic strategy as a putative mono-ADP-ribosyltransferase and a possible virulence factor from Paenibacillus larvae, which is the causative agent of American Foulbrood in honey bees. C3larvin targets RhoA as a substrate for its transferase reaction, and kinetics for both the NAD⁺ (K_m = 34 ± 12 μm) and RhoA (K_m = 17 ± 3 μm) substrates were characterized for this enzyme from the mono-ADP-ribosyltransferase C3 toxin subgroup. C3larvin is toxic to yeast when expressed in the cytoplasm, and catalytic variants of the enzyme lost the ability to kill the yeast host, indicating that the toxin exerts its lethality through its enzyme activity. A small molecule inhibitor of C3larvin enzymatic activity was discovered called M3 (K_i = 11 ± 2 μm), and to our knowledge, is the first inhibitor of transferase activity of the C3 toxin family. C3larvin was crystallized, and its crystal structure (apoenzyme) was solved to 2.3 Å resolution. C3larvin was also shown to have a different mechanism of cell entry from other C3 toxins.     

Isolation,cDNA Cloning,and Structure-based Functional Characterization of Oryctin,a Hemolymph Protein from the Coconut Rhinoceros Beetle,Oryctes rhinoceros,as a Novel Serine Protease Inhibitor

We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant ¹³C,¹⁵N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with K_i values of 3.9 × 10⁻¹⁰ m, 6.2 × 10⁻¹⁰ m, 1.4 × 10⁻⁹ m, and 1.2 × 10⁻⁸ m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections.     

Estrogen Modification of Human Glutamate Dehydrogenases Is Linked to Enzyme Activation State

Mammalian glutamate dehydrogenase (GDH) is a housekeeping enzyme central to the metabolism of glutamate. Its activity is potently inhibited by GTP (IC₅₀ = 0.1–0.3 μm) and thought to be controlled by the need of the cell in ATP. Estrogens are also known to inhibit mammalian GDH, but at relatively high concentrations. Because, in addition to this housekeeping human (h) GDH1, humans have acquired via a duplication event an hGDH2 isoform expressed in human cortical astrocytes, we tested here the interaction of estrogens with the two human isoenzymes. The results showed that, under base-line conditions, diethylstilbestrol potently inhibited hGDH2 (IC₅₀ = 0.08 ± 0.01 μm) and with ∼18-fold lower affinity hGDH1 (IC₅₀ = 1.67 ± 0.06 μm; p < 0.001). Similarly, 17β-estradiol showed a ∼18-fold higher affinity for hGDH2 (IC₅₀ = 1.53 ± 0.24 μm) than for hGDH1 (IC₅₀ = 26.94 ± 1.07 μm; p < 0.001). Also, estriol and progesterone were more potent inhibitors of hGDH2 than hGDH1. Structure/function analyses revealed that the evolutionary R443S substitution, which confers low basal activity, was largely responsible for sensitivity of hGDH2 to estrogens. Inhibition of both human GDHs by estrogens was inversely related to their state of activation induced by ADP, with the slope of this correlation being steeper for hGDH2 than for hGDH1. Also, the study of hGDH1 and hGDH2 mutants displaying different states of activation revealed that the affinity of estrogen for these enzymes correlated inversely (R = 0.99; p = 0.0001) with basal catalytic activity. Because astrocytes are known to synthesize estrogens, these hormones, by interacting potently with hGDH2 in its closed state, may contribute to regulation of glutamate metabolism in brain.     

Amino ketone formation and aminopropanol-dehydrogenase activity in rat-liver preparations

1. Rat tissue homogenates convert dl-1-aminopropan-2-ol into aminoacetone. Liver homogenates have relatively high aminopropanol-dehydrogenase activity compared with kidney, heart, spleen and muscle preparations. 2. Maximum activity of liver homogenates is exhibited at pH9·8. The K_m for aminopropanol is approx. 15mm, calculated for a single enantiomorph, and the maximum activity is approx. 9mμmoles of aminoacetone formed/mg. wet wt. of liver/hr.at 37°. Aminoacetone is also formed from l-threonine, but less rapidly. An unidentified amino ketone is formed from dl-4-amino-3-hydroxybutyrate, the K_m for which is approx. 200mm at pH9·8. 3. Aminopropanol-dehydrogenase activity in homogenates is inhibited non-competitively by dl-3-hydroxybutyrate, the K_i being approx. 200mm. EDTA and other chelating agents are weakly inhibitory, and whereas potassium chloride activates slightly at low concentrations, inhibition occurs at 50–100mm. 4. It is concluded that aminopropanol-dehydrogenase is located in mitochondria, and in contrast with l-threonine dehydrogenase can be readily solubilized from mitochondrial preparations by ultrasonic treatment. 5. Soluble extracts of disintegrated mitochondria exhibit maximum aminopropanol-dehydrogenase activity at pH9·1 At this pH, K_m values for the amino alcohol and NAD⁺ are approx. 200 and 1·3mm respectively. Under optimum conditions the maximum velocity is approx. 70mμmoles of aminoacetone formed/mg. of protein/hr. at 37°. Chelating agents and thiol reagents appear to have little effect on enzyme activity, but potassium chloride inhibits at all concentrations tested up to 80mm. dl-3-Hydroxybutyrate is only slightly inhibitory. 6. Dehydrogenase activities for l-threonine and dl-4-amino-3-hydroxybutyrate appear to be distinct from that for aminopropanol. 7. Intraperitoneal injection of aminopropanol into rats leads to excretion of aminoacetone in the urine. Aminoacetone excretion proportional to the amount of the amino alcohol administered, is complete within 24hr., but represents less than 0·1% of the dose given. 8. The possible metabolic role of amino alcohol dehydrogenases is discussed.     

β-Galactosidase-catalysed hydrolysis of β-d-galactopyranosyl azide

1. β-d-Galactopyranosyl azide is hydrolysed by the β-galactosidase of Escherichia coli to galactose and azide ion at a mechanistically significant rate. 2. Methyl 1-thio-β-d-galactopyranoside is a competitive inhibitor of the hydrolysis of the azide and of o-nitrophenyl β-d-galactopyranoside with K_i 1.8mm. 3. β-Galactosidase can thus hydrolyse a range of substrates of general structure β-d-galactopyranosyl-X(Y), where the atom X has a lone pair of electrons on which the enzyme may act as a Lewis or Brønsted acid, but in which the length of the bond cleaved varies significantly, which is inconsistent with the orbital steering hypothesis.     

Convergent Evolution in the Assembly of Polyubiquitin Degradation Signals by the Shigella flexneri IpaH9.8 Ligase

The human pathogen Shigella flexneri subverts host function and defenses by deploying a cohort of effector proteins via a type III secretion system. The IpaH family of 10 such effectors mimics ubiquitin ligases but bears no sequence or structural homology to their eukaryotic counterpoints. Using rates of ¹²⁵I-polyubiquitin chain formation as a functional read out, IpaH9.8 displays V-type positive cooperativity with respect to varying concentrations of its Ubc5B∼¹²⁵I-ubiquitin thioester co-substrate in the nanomolar range ([S]_½ = 140 ± 32 nm; n = 1.8 ± 0.1) and cooperative substrate inhibition at micromolar concentrations ([S]_½ = 740 ± 240 nm; n = 1.7 ± 0.2), requiring ordered binding to two functionally distinct sites per subunit. The isosteric substrate analog Ubc5BC85S-ubiquitin oxyester acts as a competitive inhibitor of wild-type Ubc5B∼¹²⁵I-ubiquitin thioester (K_i = 117 ± 29 nm), whereas a Ubc5BC85A product analog shows noncompetitive inhibition (K_i = 2.2 ± 0.5 μm), consistent with the two-site model. Re-evaluation of a related IpaH3 crystal structure (PDB entry 3CVR) identifies a symmetric dimer consistent with the observed cooperativity. Genetic disruption of the predicted IpaH9.8 dimer interface reduces the solution molecular weight and significantly ablates the k_cat but not [S]_½ for polyubiquitin chain formation. Other studies demonstrate that cooperativity requires the N-terminal leucine-rich repeat-targeting domain and is transduced through Phe³⁹⁵. Additionally, these mechanistic features are conserved in a distantly related SspH2 Salmonella enterica ligase. Kinetic parallels between IpaH9.8 and the recently revised mechanism for E6AP/UBE3A (Ronchi, V. P., Klein, J. M., and Haas, A. L. (2013) E6AP/UBE3A ubiquitin ligase harbors two E2∼ubiquitin binding sites. J. Biol. Chem. 288, 10349–10360) suggest convergent evolution of the catalytic mechanisms for prokaryotic and eukaryotic ligases.     

Terminal Oxidases of Chlorella pyrenoidosa

In studies of the kinetics of oxygen uptake by glucose-stimulated Chlorella pyrenoidosa, two terminal oxidases could be distinguished. The cytochrome oxidase of Chlorella has a Km (O₂) of 2.1 ± 0.3 μm, while the second oxidase has a Km (O₂) of 6.7 ± 0.5 μm, and a maximum capacity about one-quarter of that of the cytochrome system. The identity of the second oxidase is unknown, but it is not inhibited by carbon monoxide, 1 mm cyanide, 0.1 mm thiocyanate, or 1 mm 8-hydroxyquinoline. In fresh cultures, the second oxidase accounts for at most 35% of the total oxygen uptake.     

The activities and kinetic properties of purine phosphoribosyltransferases in developing mouse liver

1. The total activity of adenine phosphoribosyltransferase/liver of mice remained constant from 1 to 16 days after birth despite a fourfold increase in liver weight. The total activity of this enzyme increased fivefold from 16 to 36 days and then remained relatively constant at least until 96 days after birth. Total hypoxanthine-phosphoribosyltransferase activity/liver steadily increased between 1 and 57 days after birth. 2. The mean K_m of 5-phosphoribosyl pyrophosphate with adenine phosphoribosyltransferase was 10·1μm between 3 and 11 days, at 64 days and at 96 days after birth. Between 17 and 51 days the mean K_m value was 3·0μm. The K_m of 5-phosphoribosyl pyrophosphate with hypoxanthine phosphoribosyltransferase remained constant at 28·2μm between 2 and 64 days. 3. Adenine-phosphoribosyltransferase activity was stimulated between 15 and 83% by 60μm-ATP when extracts were made between 3 and 11 days, at 64 days or at 96 days after birth. Between 17 and 51 days ATP had little stimulatory effect on the activity of this enzyme. 4. AMP competed with 5-phosphoribosyl pyrophosphate in the reaction catalysed by adenine phosphoribosyltransferase. Liver extracts containing enzyme with a low value of K_m for 5-phosphoribosyl pyrophosphate (3μm) had a K_m/K_i ratio approximately half that of extracts with a high value of K_m (10μm). 5. The results indicate that two different forms of adenine phosphoribosyltransferase can exist in mouse liver at different stages of development. The physiological significance of these findings is discussed.     

A New Group of Aromatic Prenyltransferases in Fungi,Catalyzing a 2,7-Dihydroxynaphthalene 3-Dimethylallyl-transferase Reaction

Five fungal genomes from the Ascomycota (sac fungi) were found to contain a gene with sequence similarity to a recently discovered small group of bacterial prenyltransferases that catalyze the C-prenylation of aromatic substrates in secondary metabolism. The genes from Aspergillus terreus NIH2624, Botryotinia fuckeliana B05.10 and Sclerotinia sclerotiorum 1980 were expressed in Escherichia coli, and the resulting His₈-tagged proteins were purified and investigated biochemically. Their substrate specificity was found to be different from that of any other prenyltransferase investigated previously. Using 2,7-dihydroxynaphthalene (2,7-DHN) and dimethylallyl diphosphate as substrates, they catalyzed a regiospecific Friedel-Crafts alkylation of 2,7-DHN at position 3. Using the enzyme of A. terreus, the K_m values for 2,7-DHN and dimethylallyl diphosphate were determined as 324 ± 25 μm and 325 ± 35 μm, respectively, and k_cat as 0.026 ± 0.001 s⁻¹. A significantly lower level of prenylation activity was found using dihydrophenazine-1-carboxylic acid as aromatic substrate, and only traces of products were detected with aspulvinone E, flaviolin, or 4-hydroxybenzoic acid. No product was formed with l-tryptophan, l-tyrosine, or 4-hydroxyphenylpyruvate. The genes for these fungal prenyltransferases are not located within recognizable secondary metabolic gene clusters. Their physiological function is yet unknown.     

Structure and Function of Human Xylulokinase,an Enzyme with Important Roles in Carbohydrate Metabolism

d-Xylulokinase (XK; EC 2.7.1.17) catalyzes the ATP-dependent phosphorylation of d-xylulose (Xu) to produce xylulose 5-phosphate (Xu5P). In mammals, XK is the last enzyme in the glucuronate-xylulose pathway, active in the liver and kidneys, and is linked through its product Xu5P to the pentose-phosphate pathway. XK may play an important role in metabolic disease, given that Xu5P is a key regulator of glucose metabolism and lipogenesis. We have expressed the product of a putative human XK gene and identified it as the authentic human d-xylulokinase (hXK). NMR studies with a variety of sugars showed that hXK acts only on d-xylulose, and a coupled photometric assay established its key kinetic parameters as K_m(Xu) = 24 ± 3 μm and k_cat = 35 ± 5 s⁻¹. Crystal structures were determined for hXK, on its own and in complexes with Xu, ADP, and a fluorinated inhibitor. These reveal that hXK has a two-domain fold characteristic of the sugar kinase/hsp70/actin superfamily, with glycerol kinase as its closest relative. Xu binds to domain-I and ADP to domain-II, but in this open form of hXK they are 10 Å apart, implying that a large scale conformational change is required for catalysis. Xu binds in its linear keto-form, sandwiched between a Trp side chain and polar side chains that provide exquisite hydrogen bonding recognition. The hXK structure provides a basis for the design of specific inhibitors with which to probe its roles in sugar metabolism and metabolic disease.     

Mechanism of Endogenous Regulation of the Type I Interferon Response by Suppressor of IκB Kinase ? (SIKE), a Novel Substrate of TANK-binding Kinase 1 (TBK1)

TANK-binding kinase 1 (TBK1) serves as a key convergence point in multiple innate immune signaling pathways. In response to receptor-mediated pathogen detection, TBK1 phosphorylation promotes production of pro-inflammatory cytokines and type I interferons. Increasingly, TBK1 dysregulation has been linked to autoimmune disorders and cancers, heightening the need to understand the regulatory controls of TBK1 activity. Here, we describe the mechanism by which suppressor of IKKϵ (SIKE) inhibits TBK1-mediated phosphorylation of interferon regulatory factor 3 (IRF3), which is essential to type I interferon production. Kinetic analyses showed that SIKE not only inhibits IRF3 phosphorylation but is also a high affinity TBK1 substrate. With respect to IRF3 phosphorylation, SIKE functioned as a mixed-type inhibitor (K_{i, app} = 350 nm) rather than, given its status as a TBK1 substrate, as a competitive inhibitor. TBK1 phosphorylation of IRF3 and SIKE displayed negative cooperativity. Both substrates shared a similar K_m value at low substrate concentrations (∼50 nm) but deviated >8-fold at higher substrate concentrations (IRF3 = 3.5 μm; SIKE = 0.4 μm). TBK1-SIKE interactions were modulated by SIKE phosphorylation, clustered in the C-terminal portion of SIKE (Ser-133, -185, -187, -188, -190, and -198). These sites exhibited striking homology to the phosphorylation motif of IRF3. Mutagenic probing revealed that phosphorylation of Ser-185 controlled TBK1-SIKE interactions. Taken together, our studies demonstrate for the first time that SIKE functions as a TBK1 substrate and inhibits TBK1-mediated IRF3 phosphorylation by forming a high affinity TBK1-SIKE complex. These findings provide key insights into the endogenous control of a critical catalytic hub that is achieved not by direct repression of activity but by redirection of catalysis through substrate affinity.     

NAD-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

The NAD⁺-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP⁺-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD⁺ and NADP⁺-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD⁺-dependent isocitrate dehydrogenase showed sigmodial kinetics in response to isocitrate (S_0.5 = 0.3 mm). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD⁺ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD⁺ (K_m = 0.2 mm). NADH was a competitive inhibitor (K_i = 0.2 mm) and, unexpectedly, NADPH was a noncompetitive inhibitor (K_i = 0.3 mm). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.     

Substrate and inhibitor specificity of the cholesterol oxidase in bovine adrenal cortex

1. Cholesteryl 3β-sulphate is oxidized in vitro by preparations of bovine adrenal-cortex mitochondria to pregnenolone sulphate and isocaproic acid (4-methyl-pentanoic acid) without hydrolysis of the ester linkage. 2. Free cholesterol is the preferred substrate for adrenal-cortex cholesterol oxidase; the apparent K_m for cholesteryl sulphate is 500μm and for free cholesterol 50μm under the same conditions. 3. Cholesteryl 3β-acetate is hydrolysed by bovine adrenal-cortex mitochondria in vitro to free cholesterol, which is subsequently oxidized to more polar steroids and isocaproic acid. Evidence was obtained that other cholesterol esters behave similarly. Cholesterol esters may thus act as precursors of steroid hormones. 4. Cholest-4-en-3-one is only poorly oxidized to isocaproic acid and more polar steroids and thus is probably not a significant precursor of steroid hormones. 5. Cholesteryl esters inhibit the oxidation of cholesterol competitively (K_i for cholesteryl phosphate 28μm, for cholesteryl sulphate 110μm, for cholesteryl acetate 65μm) but pregnenolone esters do not inhibit this system. 6. Pregnenolone and 20α-hydroxycholesterol (both metabolites of cholesterol in this system) inhibit the oxidation of cholesterol non-competitively. K_i for pregnenolone is 130μm and K_i for 20α-hydroxycholesterol is 17μm. 7. 25-Oxo-27-norcholesterol inhibits cholesterol oxidation non-competitively (K_i16μm). A number of other Δ⁵-3β-hydroxy steroids inhibit cholesterol oxidation and evidence was obtained that the 3β-hydroxyl group was necessary for inhibitory activity. 8. Pregnenolone, 20α-hydroxycholesterol and 25-oxo-27-norcholesterol inhibit oxidation of cholesteryl sulphate by this system but their sulphates do not. 9. 3β-Hydroxychol-5-enoic acid, 3α-hydroxy-5β-cholanic acid and 3β-hydroxy-22,23-bisnorchol-5-enoic acid stimulated formation of isocaproic acid from cholesterol. 10. No evidence was obtained that phosphorylation or sulphation are obligatory steps in cholesterol oxidation by adrenal-cortex mitochondria. 11. The cholesteryl 3β-sulphate sulphatase of bovine adrenal cortex was found mostly in the microsomal fraction and was inhibited by inorganic phosphate.     

The metabolism of cyclic nucleotides in the guinea-pig pancreas. Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase

Both cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase were recovered mainly from the supernatant fractions of guinea-pig pancreas, but a higher proportion of the activity of the former was associated with the pellet fractions. The activities in the supernatant were not separated by gel filtration, but were clearly separated by subsequent chromatography on an anion-exchange resin. The activities of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase had high-affinity (K_m 6.5±1.1μm and 31.9±3.9μm respectively) and low-affinity (K_m 0.56±0.05mm and 0.32±0.03mm respectively) components. The activity of neither enzyme was affected by the pancreatic secretogens, cholecystokinin-pancreozymin, secretin and carbachol. Removal of ions by gel filtration resulted in a marked reduction in cyclic nucleotide phosphodiesterase activity, which could be restored by addition of Mg²⁺. Mn²⁺ (3mm) was as effective as Mg²⁺ (3mm) in the case of cyclic AMP phosphodiesterase, but was less than half as effective in the case of cyclic GMP phosphodiesterase. The metal-ion chelators, EDTA and EGTA, also decreased activity. Ca²⁺ (1mm) did not affect the activity of cyclic nucleotide phosphodiesterase when the concentration of Mg²⁺ was 3mm. At concentrations of Mg²⁺ between 0.1 and 1mm, 1mm-Ca²⁺ was activatory, and at concentrations of Mg²⁺ below 0.1mm, 1mm-Ca²⁺ was inhibitory. These results are discussed in terms of the possible significance of cyclic nucleotide phosphodiesterase in the physiological control of cyclic nucleotide concentrations during stimulus–secretion coupling.     

The enzymes forming isopentenyl pyrophosphate from 5-phosphomevalonate (mevalonate 5-phosphate) in the latex of Hevea brasiliensis

1. Phosphomevalonate kinase and 5-pyrophosphomevalonate decarboxylase have been purified from the freeze-dried latex serum of the commercial rubber tree Hevea brasiliensis. 2. The phosphomevalonate kinase was acid- and heat-labile and required the presence of a thiol to maintain activity. 3. The 5-pyrophosphomevalonate decarboxylase was relatively acid-stable and more heat-stable than the phosphokinase. 4. Maximum activity of the phosphokinase was achieved at pH 7.2 with 0.2mm-5-phosphomevalonate (K_m 0.042mm), 2.0mm-ATP (K_m 0.19mm) and 8mm-Mg²⁺ at 40°C. The apparent activation energy was 14.8kcal/mol. 5. Maximum activity of 5-pyrophosphomevalonate decarboxylase was achieved at pH5.5–6.5 with 0.1mm-5-pyrophosphomevalonate (K_m 0.004mm), 1.5mm-ATP (K_m 0.12mm) and 2mm-Mg²⁺. The apparent activation energy was 13.7kcal/mol. The enzyme was somewhat sensitive to inhibition by its products, isopentenyl pyrophosphate and ADP.     

Nitric-oxide Dioxygenase Function of Human Cytoglobin with Cellular Reductants and in Rat Hepatocytes

Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b₅ were found to support a high NOD activity. Cygb-NOD activity shows respective K_m values for ascorbate, cytochrome b₅, NO, and O₂ of 0.25 mm, 0.3 μm, 40 nm, and ∼20 μm and achieves a k_cat of 0.5 s⁻¹. Ascorbate and cytochrome b₅ reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m⁻¹ s⁻¹ and 3 × 10⁶ m⁻¹ s⁻¹, respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar k_cat of 1.2 s⁻¹, a K_m(NO) of 40 nm, and a k_cat/K_m(NO) (k′_NOD) value of 3 × 10⁷ m⁻¹ s⁻¹, demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O₂] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b₅ as reductants.