Competition by 4-chloronitrosobenzene for the active glycolaldehyde intermediate of transketolase

The incubation of 4-chloronitrosobenzene with yeast transketolase, Mg²⁺, and thiamime pyrophosphate in the presence of excess xylulose-5-phosphate resulted in the formation of N-(4-chlorophenyl)glycolhydroxamic acid. This enzyme-catalyzed C₂ transfer displayed a K_m of 0.92 mM and a V_max of 5.2 × 10^?2 μmol min^?1 unit enzyme^?1. Conversion was inhibited by the normal acceptor sugar, ribose-5-phosphate, with a K_i of 0.35 mM. Kinetic analysis showed inhibition was competitive in nature, reinforcing the proposed theory for similarity in catalytic formation of both the hydroxamic acid and sedoheptulose-7-phosphate. Most interesting about the conversion of this alternative substrate is that even at high concentrations of ribose-5-phosphate, a significant amount of the nitroso compound was converted to the hydroxamic acid, implying that 4-chloronitrosobenzene can successfully compete for active glycoaldehyde. Using the yeast enzyme as a model for transketolase in higher organisms, the adventitious conversion of such xenobiotics in vivo is proposed.     

Hydroxamic acids from the reaction of active acetaldehyde with aromatic nitroso compounds

α-Hydroxyethylthiamine (4) reacts with nitrosobenzene (5) to produce N-phenylacetohydroxamic acid (7). A mechanism for this reaction is proposed. The possibility that similar reactions can occur in vivo with the production of carcinogenic hydroxamic acids is discussed.     

The nonmicrosomal production of N-(4-chlorophenyl)-glycolhydroxamic acid from 4-chloronitrosobenzene by rat liver homogenates

The incubation of 4-chloronitrosobenzene (4-CNB) with subcellular fractions of rat liver resulted in the formation of a previously unknown type of hydroxamic acid metabolite for mammals. This new metabolite, N-(4-chlorophenyl)glycolhydroxamic acid (Gl-CHA), is most likely formed through the action of liver transketolase on the substrate 4-CNB. Gl-CHA was produced only by the 10 000g and 105 000g supernatant fractions, and required glucose-6-phosphate as an energy source. No hydroxamic acid metabolites were produced in detectable quantities by the microsomal fraction of the rat liver homogenate. Gl-CHA was positively identified by isolation and comparison to an authentic sample of Gl-CHA. Authentic Gl-CHA was prepared by the condensation of 4-chlorophenylhydroxylamine with glycolic acid in the presence of dicylohexylcarbodiimide. The highest observed conversion of 4-CNB to Gl-CHA was 18%, which occurred at the lowest concentration of 4-CNB incubated with the 105 000g supernatant. Gl-CHA was not produced by C-hydroxylation of the corresponding acetyl-derived hydroxamic acid, since none of the subcellular fractions of rat liver would effect this conversion. The incubation of 4-chloroaniline under identical conditions failed to result in the production of Gl-CHA; however, such an observation is probably not important to the possibility that Gl-CHA might be a significant metabolite in vivo.     

Synthesis, enzymatic inhibition, and cancer cell growth inhibition of novel delta-lactam-based histone deacetylase (HDAC) inhibitors

delta-Lactam-based hydroxamic acids, inhibitors of histone deacetylase (HDAC), have been synthesized via ring closure metathesis of key diene intermediates followed by conversion to hydroxamic acid analogues. The hydroxamic acids 12a, 12b, and 17c showed potent inhibitory activity in HDAC enzyme assay. The hydroxamic acid 12b exhibited growth inhibitory activity on five human tumor cell lines, showing good sensitivity on the MDA-MB-231 breast tumor cell.     

Enzymatic generation of N-[4-dimethylamino)phenyl]acetohydroxamic acid by the action of pyruvate decarboxylase on 4-(dimethylamino)nitrosobenzene

The established ability of pyruvate decarboxylase to catalyze the conversion of nitroso aromatics to hydroxamic acids was utilized to generate the previously unknown hydroxamic acid 2. Although 2 could not be isolated in pure form from enzymatic reactions, evidence for its production is presented in this study. Under the conditions of the enzymatic reaction, 2 undergoes a slower reduction to give the corresponding acetanilide 3, which was isolated and characterized. The isomeric hydroxamic acid 4 was synthesized and its stability compared to that of 2. The much greater reactivity of the hydroxamic acid 2, particularly evidenced by its facile reduction to 3, was explained on the basis of the potential for the formation of the N-acylquinonediimine cation, 9.     

Comparative aspects of hydroxamic acid production by thiamine-dependent enzymes

The reactions of 4-chloronitrosobenzene with pyruvate decarboxylase and transketolase were investigated by use of a new high-pressure liquid chromatography method to determine any differences between these two enzymes with respect to hydroxamic acid production. In addition to the previously established difference in the type of hydroxamic acid produced by the two enzymes, several new and interesting differences in their reaction with nitrosoaromatics were discovered. Most notable was the finding that pyruvate decarboxylase gave 4-chlorophenylhydroxylamine as the major product from 4-chloronitrosobenzene, while transketolase did not produce any detectable hydroxylamine. A redox mechanism was proposed to account for arylhydroxylamine production by pyruvate decarboxylase. This redox mechanism can also explain hydroxamic acid production by pyruvate decarboxylase; however, a previously proposed nucleophilic reaction mechanism occurring simultaneously could not be totally disproven. Either of the two mechanisms is equally likely for transktolase action in view of the present evidence. Another major difference between these enzymes is that the rate of 4-chloronitrosobenzene conversion was found to be much faster for pyruvate decarboxylase than for transketolase when each enzyme was subjected to its own optimal reaction conditions. Transketolase displayed typical enzyme saturation kinetics with 4-chloronitrosobenzene with a K_m of 0.31 mM and V_max of 0.033 μmol ml^?1 min^?1 unit^?1 relative to 5 mMd-fructose 6-phosphate as sugar substrate. On the other hand, the reaction with pyruvate decarboxylase was first order in 4-chloronitrosobenzene with a combined rate constant of 2.0 min^?1 unit^?1 ml.     

New reaction sequences for the non-oxidative pentose phosphate pathway.

1. Reactions leading to the formation of 14C-labelled volatile compounds and compounds volatile under acid conditions were investigated in a system actively synthesizing hexose 6-phosphates from [U-14C]ribose 5-phosphate by reactions catalysed by enzymes prepared from acetone-dried powder of rat liver; no reactions involving 14C-labelled volatile compounds were detected. Similarly the fixation of 14C-labelled volatile compounds into hexose 6-phosphate could not be detected. 2. A complete carbon balance was made for the reactants, intermediates and products of the reactions involved in the conversion of ribose 5-phosphate into hexose 6-phosphate by enzymes of rat liver. Five additional intermediates of pentose 5-phosphate metabolism in liver were detected, namely D-manno-heptulose 7-phosphate, D-altro-heptulose 1,7-bisphosphate, D-glycero-D-ido-octulose 1,8-bisphosphate, D-glycero-D-altro-octulose 1,8-bisphosphate and D-arabinose 5-phosphate. 3. D-Arabinose 5-phosphate was found to be utilized by a rat liver enzyme preparation to produce both hexose 6-phosphate and triose phosphate. 4. D-Arabinose 5-phosphate was reversibly converted into other pentose 5-phosphates. Paper chromatographic and enzymic evidence indicated that the conversion involved an enzyme tentatively named arabinose phosphate 2-epimerase, which catalyses the following reaction: D-arabinose 5-P in equilibrium D-ribose-5-P. 5. A variety of rat tissues also utilized D-arabinose 5-phosphate to produce both hexose 6-phosphate and triose phosphate and at a rate comparable with that obtained with D-ribose 5-phosphate. 6. A new reaction sequence for the non-oxidative pentose phosphate pathway in liver is proposed.     

Quantitative determination of N-arylaceto- and N-arylglycolhydroxamic acids in biochemical reaction mixtures

A high-performance liquid chromatography (hplc) solvent system was developed for use with C₁₈ bonded packings that effected the chromatographic resolution of monoaromatic hydroxamic acids in complex mixtures. The success of this hplc system resulted from the incorporation of an aliphatic hydroxamic acid additive into the elution solvent. This additive, desferal mesylate, suppressed chemisorption effects between the stationary phase of the chromatographic packing and the aromatic hydroxamic acids being analyzed. The detectability limit for acetyl-derived aromatic hydroxamic acids was about 10 ng, while that for glycolyl-derived aromatic hydroxamic acids was about 5 ng. This level of sensitivity was sufficiently low to allow for the direct detection of these components in enzymatic reaction mixutres. By manipulation of the percentage methanol in the aqueous solvent the resolution of either type of hydroxamic acid from other related metabolites was readily accomplished.     

A coupled optical enzyme assay for phosphopentomutase

Published assays for phosphopentomutase activity are based on acid lability differences between ribose 1-phosphate and ribose 5-phosphate. The present work describes a new method in which the isomerization of ribose 5-phosphate to ribose 1-phosphate is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: ribose 1-phosphate + adenine ? phosphate + adenosine (adenosine phosphorylase); adenosine + H₂O → inosine + NH₃ (adenosine deaminase). The method has been used to show some properties of Escherichia coli phosphopentomutase.     

Oxidative degradation of haemoglobin by nitrosobenzene in the erythrocyte.

Substitutions on the benzene ring of nitrosobenzene did not have the same effect on oxidative haemolysis as substitutions on phenylhydrazine. We previously found that the haemolytic effect of arylhydrazines paralleled their oxidative conversion into ligands of ferrihaemoglobin. In contrast, although most substituted nitrosobenzenes that are ligands of ferrohaemoglobin caused haemolysis and most that are not ligands failed to cause nitrosoarenes appeared to be related more closely to the ease of their reduction to arylhydroxylamines than to their properties as ligands. We propose a mechanism of oxidative degradation whereby the cyclic formation of phenylhydroxylamine from nitrosobenzene within an erythrocyte leads to the accumulation of H2O2, which then reacts with ferrohaemoglobin to initiate the oxidative cleavage of haem. The posulated active intermediate in this reaction is the same as that previously proposed in the oxidative degradation of haemoglobin by phenylhydrzine and in the coupled oxidation of ascorbic acid and haemoglobin.     

Synthesis of polyfunctional hydroxamic acids for potential use in iron chelation therapy

Two new multidentate N-methylhydroxamic acids were prepared and characterized. β-Cyclodextrin was esterified by treatment with succinic anhydride. The resulting carboxyl groups (14 per cyclodextrin) were converted to the N-hydroxysuccinimide esters and then on to the hydroxamic acids by treatment with N-methylhydroxylamine. Tetracyanoethylation of cyclohexanone followed by hydrolysis of the nitrile and conversion of the carboxylic acid to hydroxamic acid produced a tetrahydroxamic acid derivative of cyclohexanone. Infrared, ¹H NMR, and ¹³C NMR were consistent with the proposed structures. The hydroxamic acids were water soluble and formed the typical red-brown iron complexes. Stability constants (log K) of 29–30 for the iron complexes indicated a strong chelate effect. Animal tests indicated that the two compounds were only weakly effective in removing iron in vivo from iron-overloaded mice. The potency was only 0.1 that of the standard drug desferrioxamine-B.     

Simple inhibitors of histone deacetylase activity that combine features of short-chain fatty acid and hydroxamic acid inhibitors

Butyric acid and trichostatin A (TSA) are anti-cancer compounds that cause the upregulation of genes involved in differentiation and cell cycle regulation by inhibiting histone deacetylase (HDAC) activity. In this study we have synthesized and evaluated compounds that combine the bioavailability of short-chain fatty acids, like butyric acid, with the bidentate binding ability of TSA. A series of analogs were made to examine the effects of chain length, simple aromatic cap groups, and substituted hydroxamates on the compounds' ability to inhibit rat-liver HDAC using a fluorometric assay. In keeping with previous structure-activity relationships, the most effective inhibitors consisted of longer chains and hydroxamic acid groups. It was found that 5-phenylvaleric hydroxamic acid and 4-benzoylbutyric hydroxamic acid were the most potent inhibitors with IC50's of 5 microM and 133 microM respectively.     

Simple inhibitors of histone deacetylase activity that combine features of short-chain fatty acid and hydroxamic acid inhibitors

Butyric acid and trichostatin A (TSA) are anti-cancer compounds that cause the upregulation of genes involved in differentiation and cell cycle regulation by inhibiting histone deacetylase (HDAC) activity. In this study we have synthesized and evaluated compounds that combine the bioavailability of short-chain fatty acids, like butyric acid, with the bidentate binding ability of TSA. A series of analogs were made to examine the effects of chain length, simple aromatic cap groups, and substituted hydroxamates on the compounds' ability to inhibit rat-liver HDAC using a fluorometric assay. In keeping with previous structure-activity relationships, the most effective inhibitors consisted of longer chains and hydroxamic acid groups. It was found that 5-phenylvaleric hydroxamic acid and 4-benzoylbutyric hydroxamic acid were the most potent inhibitors with IC₅₀'s of 5 μM and 133 μM respectively.     

Electron spin resonance studies on the lipoxygenase reaction by spin trapping and spin labelling methods.

The rate of oxygenation and that of trapping linoleic acid free radicals in the lipoxygenase [EC 1.13.11.12] reaction were measured in the presence of linoleic acid, oxygen, and nitrosobenzene at various concentrations, with a Clark oxygen electrode and ESR spectroscopy. The results were interpreted under the assumption that the free radical of linoleic acid, an intermediate of the lipoxygenase reaction, reacts competitively with oxygen or nitrosobenzene. The oxidation of the iron in the active site of lipoxygenase caused by the spin label reagent, 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinyloxyl, was also observed by ESR- and fluorescence-spectroscopy.     

Preliminary studies on the inhibition of D-sorbitol-6-phosphate 2-dehydrogenase from Escherichia coli with substrate analogues

D-Sorbitol-6-phosphate 2-dehydrogenase catalyzes the NADH-dependent conversion of D-fructose 6-phosphate to D-sorbitol 6-phosphate and improved production and purification of the enzyme from Escherichia coli is reported. Preliminary inhibition studies of the enzyme revealed 5-phospho-D-arabinonohydroxamic acid and 5-phospho-D-arabinonate as new substrate analogue inhibitors of the F6P catalyzed reduction with IC50 values of (40 +/- 1) microM and (48 +/- 3) microM and corresponding Km/IC50 ratio values of 14 and 12, respectively. Furthermore, we report here the phosphomannose isomerase substrate D-mannose 6-phosphate as the best inhibitor of E. coli D-sorbitol-6-phosphate 2-dehydrogenase yet reported with an IC50 = 7.5 +/- 0.4 microM and corresponding Km/IC50 ratio = about 76.     

Oil biosynthesis in microsomal membrane preparations from Mortierella alpina

Microsomal membrane preparations from Mortierella alpina catalysed the conversion of sn-glycerol 3-phosphate and [(14)C]oleoyl-CoA to radioactive phosphatidic acid, diacylglycerol and triacylglycerol. Experiments with lysophosphatidic acid and [(14)C]oleoyl-CoA gave a similar pattern of radioactivity in the complex lipids. The specific activity of lysophosphatidate acyltransferase was almost eight times greater than sn-glycerol-3-phosphate acyltransferase, indicating that the first acylation step was limiting in oil assembly in the microsomal membranes. Little conversion of radioactive oleate into phosphatidylcholine occurred, suggesting that triacylglycerol assembly and its relationship to phosphatidylcholine metabolism differed to that found in oilseeds.     

The synthesis and purification of 2''-carboxy-D-arabinitol 1-phosphate, a natural inhibitor of ribulose 1,5-bisphosphate carboxylase, investigated by 31P n.m.r.

2'-Carboxy-D-arabinitol 1-phosphate (2CA1P), a natural inhibitor of ribulose 1,5-bisphosphate carboxylase was synthesized from 2'-carboxy-D-arabinitol 1,5-bisphosphate (2CABP). The selective dephosphorylation of 2CABP with either acid phosphatase or alkaline phosphatase was investigated by using 31P n.m.r. The n.m.r. spectra of the progress of the reactions indicated that both phosphatases preferentially removed the 5-phosphate from the bisphosphate. After the consumption of all of the bisphosphate, alkaline phosphatase generated a mixture of 2'-carboxy-D-arabinitol 1- and 5-monophosphates in the ratio of about 4:1, along with Pi. The enzyme also hydrolysed the monophosphates to 2'-carboxyarabinitol, thus decreasing the yield of 2CA1P further. In contrast, acid phosphatase catalysed almost quantitative conversion of 2CABP into 2CA1P, preferring to hydrolyse only the 5-phosphate. In either case, separation of the 2CA1P from Pi or other products of enzymic hydrolysis was readily accomplished by conventional ion-exchange chromatography or h.p.l.c.     

Carbon metabolism of chloroplasts in the dark: Oxidative pentose phosphate cycle versus glycolytic pathway

The conversion of U-labelled [¹⁴C]glucose-6-phosphate into other products by a soluble fraction of lysed spinach chloroplasts has been studied. It was found that both an oxidative pentose phosphate cycle and a glycolytic reaction sequence occur in this fraction. The formation of bisphosphates and of triose phosphates was ATP-dependent and occurred mainly via a glycolytic reaction sequence including a phosphofructokinase step. The conversion, of glucose-6-phosphate via the oxidative pentose phosphate cycle stopped with the formation of pentose monophosphates. This was found not to be because of a lack in transaldolase (or transketolase) activity, but because of the high concentration ratios of hexose monophosphate/pentose monophosphate used in our experiments for simulating the conditions in whole chloroplasts in the dark. Some regulatory properties of both the oxidative pentose phosphate cycle and of the glycolytic pathway were studied.Abbreviations DHAP dihydroxyacetone phosphate - GAP 3-phosphoglyceraldehyde - PGA 3-phosphoglycerate - HMP hexose monophosphates - including F6P fructose-6-phosphate - G6P glucose-6-phosphate - GIP glucose-1-phosphate - 6-PGL phosphogluconate - PMP pentose monophosphates - including R5P ribose-5-phosphate - Ru5P ribulose-5-phosphate - X5P xylulose-5-phosphate - E4P erythrose-4-phosphate - S7P sedoheptulose-7-phosphate - FBP fructose-1,6-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - RuBP ribulose-1,5-bisphosphate     

A convenient lactic dehydrogenase-coupled assay for determining pyridoxal 5′-phosphate in plasma

An assay for determining the concentration of pyridoxal 5′-phosphate in plasma from 0.4 ml whole blood is reported. The assay consists of incubating deproteinized plasma with d-serine apodehydratase from Escherichia coli in 0.5 mN-2-hydroxyethyl-piperazine-N′-3-propanesulfonic acid, pH 7.8, at 37°C for 15 min, and then determining the d-serine dehydratase activity of an aliquot of the incubation mixture. A lactic dehydrogenase-coupled assay (at 25°C) was used to measure the rate of enzymically catalyzed conversion of D-serine to pyruvate, wherein depletion of NADH was followed continuously at 340 nm. The concentration of pyridoxal 5′-phosphate in the plasma sample was estimated from the enzymic activity which is a linear function of the amount of pyridoxal 5′-phosphate present in the assay.