Pyridine Nucleotide Cofactors Research Articles

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase.

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3- null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.

Tartrate Dehydrogenase-Oxalate Complexes: Formation of a Stable Analog of a Reaction Intermediate Complex

Oxalate has been shown to form a stable complex with Mn-tartrate dehydrogenase-NADH complexes which are proposed to mimic an intermediate formed during catalytic turnover. The formation of this complex can be detected under turnover conditions, where oxalate acts as a time-dependent inhibitor, and under equilibrium conditions, where oxalate binding triggers a slow protein conformation change detectable by fluorescence spectroscopy. Both the rate constant for the change in fluorescence intensity upon oxalate binding and the magnitude of the fluorescence change show a hyperbolic dependence on oxalate concentration, The time-dependent inhibition by oxalate is not consistent with a model in which oxalate binds to enzyme-NAD; rather, it is proposed that inhibition arises from binding to enzyme-NADH. The apparent dissociation constants of oxalate from enzyme-NAD + and the enzyme-NADH complexes are 80 and 1 μM, respectively. The fluorescence changes which accompany oxalate binding are suggested to arise from a protein conformational change which serves to sequester reactants in the active site. Consistent with this hypothesis, it was observed that although some alternative pyridine nucleotide cofactors supported the multistep tartrate dehydrogenase-catalyzed net nonoxidative decarboxylation of meso-tartrate only at drastically reduced rates, none of the intermediate hydroxypyruvate was released into solution. In addition, fluorescence anisotropy measurements were conducted to investigate the mode of NADH binding.

Clues from a halophilic methanogen about aromatic amino acid biosynthesis in archaebacteria

Extensive diversity in features of aromatic amino acid biosynthesis and regulation has become recognized in eubacteria, but almost nothing is known about the extent to which such diversity exists within the archaebacteria. Methanohalophilus mahii, a methylotrophic halophilic methanogen, was found to synthesize l-phenylalanine and l-tyrosine via phenylpyruvate and 4-hydroxyphenylpyruvate, respectively. Enzymes capable of using l-arogenate as substrate were not found. Prephenate dehydrogenase was highly sensitive to feedback inhibition by l-tyrosine and could utilize either NADP+ (preferred) or NAD+ as cosubstrate. Tyrosine-pathway dehydrogenases having the combination of narrow specificity for a cyclohexadienyl substrate but broad specificity for pyridine nucleotide cofactor have not been described before. The chorismate mutase enzyme found is a member of a class which is insensitive to allosteric control. The most noteworthy character state was prephenate dehydratase which proved to be subject to multimetabolite control by feedback inhibitor (l-phenylalanine) and allosteric activators (l-tyrosine, l-tryptophan, l-leucine, l-methionine and l-isoleucine). This interlock type of prephenate dehydratase, also known to be broadly distributed among the gram-positive lineage of the eubacteria, was previously shown to exist in the extreme halophile, Halobacterium vallismortis. The results are consistent with the conclusion based upon 16S rRNA analyses that Methanomicrobiales and the extreme halophiles cluster together.

Characterization of the Cellular Reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): Subcellular Localization, Substrate Dependence, and Involvement of Mitochondrial Electron Transport in MTT Reduction

The MTT assay, which is widely used to measure cell proliferation and to screen for anticancer drugs, is based on reduction of the tetrazolium salt, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by actively growing cells to produce a blue formazan product. Despite broad acceptance of this assay, neither the subcellular localization, nor the biochemical events involved in MTT reduction are known. Mitochondrial involvement in MTT reduction has been inferred from studies with respiratory inhibitors using succinate as a substrate, but the contribution of this activity to overall cellular MTT reduction is unknown. Using the bone marrow-derived cell line, 32D, we investigated the subcellular localization of MTT reduction using succinate, NADH, and NADPH as substrates. At optimum substrate concentrations, MTT reduction by whole cell homogenates was greatest with NADH and least with succinate, which accounted for less than 10% of the combined activities. Using succinate, 96% of recoverable MTT reducing activity was in particulate fractions of the cell and 77% in the mitochondrial and light mitochondrial/lysosomal fractions. When NADH and NADPH were used as substrates, increased amounts of MTT reducing activity were associated with soluble fractions of the cell and association with mitochondrial fractions was less pronounced. To further characterize MTT reduction by the mitochondrial fraction, respiratory chain inhibitors were used to explore involvement of electron transport in MTT reduction. Succinate-dependent mitochondrial MTT reduction was inhibited by 80% with chlorpromazine, 70% by antimycin A, and 85-90% by thenoyltrifluoroacetone (TTFA), but inhibition was not observed with rotenone at ≤2 μM, Amytal, or azide. These results suggest that when succinate is used as an electron donor, 70-80% of mitochondrial MTT reduction occurs subsequent to transfer of electrons from cytochrome c to cytochrome oxidase, but prior to the point of azide inhibition. In contrast to succinate, NADPH-dependent mitochondrial MTT reduction was not affected by any of the respiratory inhibitors tested, and NADH-dependent reduction was only inhibited by chlorpromazine (40-50% at plateau concentrations). These results suggest that most cellular MTT reduction occurs outside the mitochondrial inner membrane and involves NADH and NADPH-dependent mechanisms that are insensitive to respiratory chain inhibitors. This interpretation is supported by whole cell studies in which rotenone failed to affect basal and interleukin-3-stimulated MTT reduction at times up to 4 h but strongly inhibited DNA synthesis. We conclude that most cellular reduction of MTT occurs extramitochondrially and probably involves the pyridine nucleotide cofactors NADH and NADPH.

Electrosynthesis and electroanalysis using Clostridium sporogenes

A polarographic approach was developed for studying the biochemical physiology and biotechnological utility of the proteolytic anaerobe Clostridium sporogenes. Methyl viologen-mediated reductions were assessed in both analytical and preparative modes, using a dropping mercury electrode and a mercury pool electrode, respectively. Using these assays, we observed an active MV-linked NAD(P) reductase in toluene-permeabilised cells of this organism. Since this enzyme could be of use in the regeneration of pyridine nucleotide cofactors used in enzymatic biotransformations, the kinetics and stability of the enzymatic activity were determined. Reduced MV could also be oxidised by exogenous D- or L-proline in both intact and toluene-permeabilised cells, via the rudimentary electron transport chain and proline reductase enzyme possessed by this organism. The polarographic approach provided a novel, continuous assay for proline reductase activity. The proline reductase activity was greatest in the mid-to-late exponential phase of growth in batch culture. The reductive carboxylation of acetyl phosphate to yield pyruvate was also studied in toluene-permeabilised cells, and the coulombic yield calculated. Whilst acetyl phosphate was reduced stoichiometrically, pyruvate formation accounted for only some 10–15% of the charge passed.

Electrosynthesis and electroanalysis using Clostridium sporogenes

A polarographic approach was developed for studying the biochemical physiology and biotechnological utility of the proteolytic anaerobe Clostridium sporogenes. Methyl viologen-mediated reductions were assessed in both analytical and preparative modes, using a dropping mercury electrode and a mercury pool electrode, respectively. Using these assays, we observed an active MV-linked NAD(P) reductase in toluene-permeabilised cells of this organism. Since this enzyme could be of use in the regeneration of pyridine nucleotide cofactors used in enzymatic biotransformations, the kinetics and stability of the enzymatic activity were determined. Reduced MV could also be oxidised by exogenous D- or L-proline in both intact and toluene-permeabilised cells, via the rudimentary electron transport chain and proline reductase enzyme possessed by this organism. The polarographic approach provided a novel, continuous assay for proline reductase activity. The proline reductase activity was greatest in the mid-to-late exponential phase of growth in batch culture. The reductive carboxylation of acetyl phosphate to yield pyruvate was also studied in toluene-permeabilised cells, and the coulombic yield calculated. Whilst acetyl phosphate was reduced stoichiometrically, pyruvate formation accounted for only some 10–15% of the charge passed.

Purification and properties of squirrel monkey (Saimiri sciureus) corticosteroid binding globulin.

Corticosteroid binding globulin (CBG), a serum glycoprotein which binds glucocorticoids and progestins with high affinity, is widely distributed throughout the animal world. Although its charge and size characteristics have largely been conserved across species, we found the behavior of CBG in squirrel monkey (Saimiri sciureus) serum during fractionation by polyacrylamide gel electrophoresis or Sephadex chromatography was consistent with a molecule about twice the size of that found in most species. To more fully understand the basis for this difference, we purified the protein by sequential affinity and DEAE-Sepharose chromatographies. The final product was obtained in greater than 60% yield and was found to migrate as a single homogeneous band when examined by electrophoresis at pH 8.3 in polyacrylamide gels varying total acrylamide concentration or under conditions of severe protein overload. The steroid binding specificity of the purified protein was identical with that of the protein in the starting serum. The ultraviolet absorption spectrum of the isolated CBG-steroid complexes revealed that the protein had no pyridine nucleotide cofactor or nucleic acid. Amino acid analyses showed that the composition of the squirrel monkey protein is quite similar to that of CBG molecules from other species but distinct from albumins, hemoglobin, or rabbit progesterone receptor. In contrast to the single protein band observed following electrophoresis under normal conditions, separations in the presence of sodium dodecyl sulfate (SDS) resolved the pure protein into two bands: one at 54,000 daltons and one at 57,000 daltons.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of a histidine residue at or near the NADPH binding site of enoyl reductase domain on the multifunctional fatty acid synthetase of chicken liver

Chemical modification of chicken liver fatty acid synthetase with the reagent ethoxyformic anhydride causes inactivation of the palmitate synthetase and enoyl reductase activities of the enzyme complex, but without significant effect on its β-ketoacyl reductase or β-ketoacyl dehydratase activity. The second-order rate constant of 0.2 mM −1·s −1 for loss of synthetase activity is equal to the value for enyol reductase, indicating that ethoxyformylation destroys, the ability of the enzyme to reduce the unsaturated acyl intermediate. The specificity of this reagent for histidine residues is indicated by the appearance of a 240 nm absorption band for ethoxyformic histidine corresponding to the modification of 2.1 residues per enzyme dimer, and by the observation that the modified enzyme is readily reactivated by hydroxylamine. A pK value of 7.1 obtained by studies of the pH rate-profile of inactivation is consistent with that of histidine. Moreover, inactivation by ethoxyformic anhydride is unaffected by reversely blocking essential SH groups of the enzyme with 5,5′-dithiobis(2-nitrobenzoic acid), and therefore does not involve the reaction of these groups. The reaction of tyrosyl groups is excluded by an unchanged absorption at 278 nm. In other experiments, it was shown that inactivation of synthetase is protected by pyridine nucleotide cofactors and nucleotide analogs containing a 2′-phosphate group, and is accompanied by the loss of 2.4 NADPH binding sites. These results implicate the presence of a histidine residue at or near the binding site for 2′-phosphate group of pyridine nucleotide in the enoyl reductase domain of the synthetase.

Alcohol dehydrogenases in Acinetobacter sp. strain HO1-N: role in hexadecane and hexadecanol metabolism.

Multiple alcohol dehydrogenases (ADH) were demonstrated in Acinetobacter sp. strain HO1-N. ADH-A and ADH-B were distinguished on the basis of electrophoretic mobility, pyridine nucleotide cofactor requirement, and substrate specificity. ADH-A is a soluble, NAD-linked, inducible ethanol dehydrogenase (EDH) exhibiting an apparent Km for ethanol of 512 microM and a Vmax of 138 nmol/min. An ethanol-negative mutant (Eth1) was isolated which contained 6.5% of wild-type EDH activity and was deficient in ADH-A. Eth1 exhibited normal growth on hexadecane and hexadecanol. A second ethanol-negative mutant (Eth3) was acetaldehyde dehydrogenase (ALDH) deficient, having 12.5% of wild-type ALDH activity. Eth3 had threefold-higher EDH activity than the wild-type strain. ALDH is a soluble, NAD-linked, ethanol-inducible enzyme which exhibited an apparent Km for acetaldehyde of 50 microM and a Vmax of 183 nmol/min. Eth3 exhibited normal growth on hexadecane, hexadecanol, and fatty aldehyde. ADH-B is a soluble, constitutive, NADP-linked ADH which was active with medium-chain-length alcohols. Hexadecanol dehydrogenase (HDH), a soluble and membrane-bound, NAD-linked ADH, was induced 5- to 11-fold by growth on hexadecane or hexadecanol. HDH exhibited apparent Kms for hexadecanol of 1.6 and 2.8 microM in crude extracts derived from hexadecane- and hexadecanol-grown cells, respectively. HDH was distinct from ADH-A and ADH-B, since HDH and ADH-A were not coinduced; Eth1 had wild-type levels of HDH; and HDH requires NAD, while ADH-B requires NADP. NAD- and NADP-independent HDH activity was not detected in the soluble or membrane fraction of extracts derived from hexadecane- or hexadecanol-grown cells. NAD-linked HDH appears to possess a functional role in hexadecane and hexadecanol dissimilation.

Specific and nonspecific components in the oxidative metabolism of estradiol by the male rat brain in vitro

The oxidative transformation of estradiol at the C-2 and C-4 positions in the male rat brain was studied by the transfer of 3H into water from [2-3H]- and [4-3H]-estradiol. The nonenzymatic transformation at either site was linear with substrate concentration. The addition of pyridine nucleotide cofactors had no effect on the transformation at the C-4 site. The C-2 reaction was greatly enhanced by NADPH or NADH and exhibited the greatest velocity in the microsomal formation in the presence of NADH. The C-2 reaction exhibited saturation with respect to substrate and NADH with apparent KM of 1.82 microM and 0.34 mM respectively and Vmax of 81.3-76.3 pmol/mg protein/60 min. The reaction at C-4 failed to exhibit substrate saturation up to 30 microM. These data indicate that male rat brain estrogen hydroxylase is specific for the C-2 position, is NADH dependent and thus different from the corresponding liver enzyme. The reaction at C-4 in the brain tissues fails to exhibit enzymatic characteristics and the formation of 4-hydroxyestrogens in that tissue may be artifactual.

Uterus of the Rat During Early Progestation: Pyridine Nucleotide Activity and Its Relation to Preimplantation Changes in Pentose Cycle Activity1

Using a radiometric enzymatic recycling method, measures were made of nicotinamide adenine dinucleotide phosphate (NADP) in the endometrium of the uterus of the adult rat during the estrous cycle and the first 6 days of pseudopregnancy. Though the content of NADP/endometrium varied between 174 pmoles (metestrus) and 1031 pmoles (proestrus) during the cycle, no significant changes in the concentration of the cofactor were measured (14–19 pmoles/mg tissue). By contrast, both the content and concentration of NADP increased between Days 1–4 of progestation. Following a decline in cofactor concentration between proestrus and Day 1 (to 9 pmoles/mg), activity increased to a sustained peak of about 24 pmoles/mg between 2400 h on Day 2 and 2400 h on Day 4. Calculation of the ratio of NADP+/NADPH revealed no significant changes during the cycle, whereas during pseudopregnancy two peaks were observed: from 0.15 (proestrus) to 0.33 (Day 1), and from 0.15 (Day 2) to 0.29 during the Day 3–4 interval. Changes in NADP concentration and in the oxidized:reduced ratio during early pseudopregnancy coincided with two previously established peaks in activity of the hexose monophosphate path(HMP). Thus, the data lend strong support to an earlier proposal that the regulation of HMP activity in the uterus during early progestation is dependent on the availability of the pyridine nucleotide cofactor and not on the relatively stable activity of the enzymes in the initial steps of this path.

Effect of chronic alcohol ingestion on the biosynthesis of steroids in rat testicular homogenate in vitro.

The activity and kinetics of delta 5-3 beta-dehydrogenase and 17 alpha-hydroxylase, using labeled pregnenolone as substrate, were measured in gonadal homogenates from rats fed alcohol or isocalorically substituted carbohydrate for 40 days. There was no difference in the rate or reaction kinetics for either enzyme noted between control and alcohol-treated animals when the assays were carried out in the presence of saturating amounts of exogenous pyridine nucleotide cofactors. However, when exogenous cofactors were omitted from the reaction mixture, there was decreased activity of the delta 5-3 beta-dehydrogenase system and increased activity of the 17 alpha-hydroxylase reaction. Furthermore, a cofactor-specific inhibiting effect on delta 5-3 beta-dehydrogenase activity by NADH and NADPH was found. Incubation of gonadal homogenates from the alcohol-treated animals with pyruvate on lactate (in the absence of exogenous cofactors) resulted in an increase and a decrease, respectively, in enzyme activity. These studies indicate that chronic alcohol use decreases gonadal delta 5-3 beta-dehydrogenase activity and that this is most likely due to an effect of the agent on the concentration and/or availability of pyridine nucleotide cofactors rather than to a direct effect on the enzyme. This phenomenon may account for the mechanism by which alcohol decreases testosterone secretion in these animals.

Pyridine nucleotide cofactor requirements of indicine N-oxide reduction by hepatic microsomal cytochrome P-450

Indicine N-oxide is reduced to indicine under anaerobic conditions by rat hepatic microsomal fraction in the presence of either NADH or NADPH. CO completely inhibits reduction, indicating the involvement of cytochrome P-450. In contrast to cytochrome P-450-catalyzed oxidations, for which NADH is about 15 per cent as effective as NADPH, NADH is 80 per cent as effective as NADPH in supporting indicine N-oxide reduction. In the presence of 3 mM NADH, the K m for indicine N-oxide is 0.37 mM, and the V max is 2.65 nmoles indicine formed/min/mg; with 3 mM NADPH the K m is 0.51 mM, and the V max is 3.00 nmoles/min/mg. NADH- and NADPH-dependent indicine N-oxide reduction appear to involve different pathways. NADH-supported reduction is inhibited 48 per cent by 0.5 mM KCN and 45 per cent by 0.8 M acetone, while NADPH-supported reduction is inhibited 3 per cent by 0.5 mM KCN and stimulated 28 per cent by 0.8 M acetone. The ability of NADH and NADPH at saturating concentrations to support indicine N-oxide reduction is additive, although this effect is not seen with phenobarbital- or 3-methylcholanthrene-pretreated animals. Phenobarbital pretreatment produces a selective increase in the V max for NADPH-dependent reduction, to 5.75 nmoles/min/mg, but has no significant effect upon K tm with NADPH or upon either the K m for the f max for NADH-supported indicine N-oxide reduction. Pretreatment with 3-methylcholanthrene has no significant effect upon the K m or V max for NADPH- or NADH-supported reduction. A possible explanation for the observations is a form of cytochrome P-450 which can accept electrons from NADH and catalyze indicine N-oxide reduction but which does not contribute to oxidative microsomal drug metabolism

Reduction of benzo(a)pyrene mutagenicity by dihydrodiol dehydrogenase.

THE enigma of how inert chemicals can exert potent mutagenic, carcinogenic, allergenic and cytotoxic effects has been much debated. It has been learned that such compounds are metabolically converted to chemically reactive species1. In the case of aromatic or olefinic compounds, monooxygenases located in the membranes of the cell can transform these compounds into epoxides2–5 which by virtue of electrophilic reactivity can bind chemically to cellular macromolecules such as DNA, RNA and proteins, thereby disturbing biochemical control mechanisms and leading to the above mentioned toxic effects. The same membranes in which such epoxides are produced possess an enzyme, epoxide hydratase, which is capable of inactivating epoxides2–4. However, with polycyclic hydrocarbons the situation is more complicated. If the epoxide groups are located on a benzo ring opposite a bay region, the resulting dihydrodiols are precursors of even more reactive dihydrodiol epoxides which readily bind to DNA6, are often strongly mutagenic7–10 and may be mainly responsible for carcinogenic effects of some polycyclic hydrocarbons11. Because, with the environmental carcinogen benzo(a)pyrene, epoxide hydratase was unable to inactivate these dihydrodiol epoxides12,13, we have investigated the ability of a dihydrodiol dehydrogenase14–16 to sequester the precursor dihydrodiols and thereby afford protection. To this end we have used bacterial mutagenesis17 as the biological endpoint to investigate the metabolic activation of benzo(a)pyrene by microsomal monooxygenase. Unfortunately, the dihydrodiol dehydrogenase requires the same pyridine nucleotide cofactor as the monooxygenases, so that simple removal or addition of cofactors could not be used to study the role of this enzyme. Therefore purification of the enzyme was necessary. We show here that in metabolic situations where epoxide hydratase cannot protect against benzo(a)pyrene metabolite-evoked mutagenicity, pure dihydrodiol dehydrogenase reduces it. This demonstrates another factor which could contribute to differences in susceptibility to polycyclic hydrocarbon-mediated toxicity between various animal species, strains, sexes, organs and developmental stages, and to differences in potency of polycyclic compounds. This enzyme also provides an additional tool for studying the molecular mechanisms of chemical carcinogenesis.

Microsomal C-nitroso reductase activity

Washed microsomes prepared from animal tissues possess the capacity to reduce 1-nitrosoadamantane to N-hydroxy-1-aminoadamantane in the presence of an NADPH generating system under aerobic conditions. No reduction of N-hydroxy-1-aminoadamantane to 1-aminoadamantane (amantadine) or of 2-adamantanone to 2-adamantanol occurred under these conditions. Reduced pyridine nucleotide cofactor is needed for the metabolic reaction. Maximum activity was obtained using washed microsomes from rabbit liver. The results obtained imply that the microsomal reduction of a C-nitroso compound involves a different enzyme system than that of a ketone reduction.

Preliminary characterization of the delta-9 desaturase of Tetrahymena pyriformis W

1. 1. In vitro assay conditions have been defined for measurement of Δ 9 desaturase activity in Tetrahymena pyriformis W. 2. 2. The reaction depends on the presence of oxygen and a reduced pyridine nucleotide cofactor. FAD supports a low level of enzymatic activity. 3. 3. Both stearyl-CoA and palmityl-CoA are acceptable substrates. Oleate formation is maximal at 30°C. 4. 4. Delta-9 desaturase activity appears to be localized in the microsomal fraction. Delta-6 and/or Δ 12 desaturase activities have also been observed. 5. 5. When the specificity of the Δ 9 desaturase towards stearyl-CoA and palmityl-CoA was observed at 30 and 16°C it was found that lowering the assay temperature did not affect specificity. Stearyl-CoA was more readily desaturated at both temperatures. 6. 6. Exogenous oleyl-CoA and diisopropylfluorophosphate had little effect on Δ 9 desaturase activity. However, cyanide strongly inhibited desaturation and a sensitivity to sulfhydryl-binding reagents has also been demonstrated.

Relation of yolk sac membrane kynurenine formamidase inhibition to certain teratogenic effects of organophosphorus insecticides and of carbaryl and eserine in chicken embryos

One type of teratogenic effect induced in chicken embryos by a variety of organophosphorus (OP) and mcthylcarbamate (MC) insecticides is associated with lowered embryo NAD levels. This appears to result from inhibition of kynurenine formamidase, which impairs conversion of tryptophan to essential pyridine nucleotide cofactors. The yolk sac membrane and the embryonic liver contain two types of kynurenine formamidase, one sensitive and the other relatively insensitive to OP and MC inhibitors. The eserine-sensitive enzyme accounts for ~ 26 percent of the overall activity for hydrolysis of N 1N x- diformyl- l-kynurenine . In studies with twenty-one OP insecticides and two MC compounds injected at day 4 of incubation, the inhibition of the eserine-sensitive kynurenine formamidase at day 9 of incubation correlates well with the lowering of NAD levels at day 12 of incubation and the intensity of teratogenic signs. Teratogens of this type combine high potency for inhibiting the relevant kynurenine formamidase in the yolk sac membrane early in development, and possibly in the liver at later stages, with adequate in tiro stability to maintain the inhibition through critical stages of embryogenesis. Thus, kynurenine formamidase joins acetylcholinesterase in the list of OP- and MC-sensitive enzymes of critical physiological significance.

Mechanism of pigeon liver malic enzyme: kinetics, specificity, and half-site stoichiometry of the alkylation of a cysteinyl residue by the substrate-inhibitor bromopyruvate.

Malic enzyme from pigeon liver is alkylated by the substrate analogue bromopyruvate, resulting in the concomitant loss of its oxidative decarboxylase and oxalacetate decarboxylase activities, but not its ability to reduce alpha-keto acids. The inactivation of oxidative decarboxylase activity follows saturation kinetics, indicating the formation of an enzyme-bromopyruvate complex (K congruent to 8 mM) prior to alkylation. The inactivation is inhibited by metal ions and pyridine nucleotide cofactors. Protection of malic enzyme by the substrates L-malate and pyruvate and the inhibitors tartronate and oxalate requires the presence of the above cofactors, which tighten the binding of these carboxylic acids in accord with the ordered kinetic scheme (Hsu, R. Y., Lardy, H. A., and Cleland, W. W. (1967), J. Biol. Chem. 242, 5315-5322). Bromopyruvate is reduced to L-bromolactate by malic enzyme and is an effective inhibitor of L-malate and pyruvate in the overall reaction. The apparent kinetic constants (90 muM-0.8 mM) are one to two orders of magnitude lower than the half-saturation constant (K) of inactivation, indicating a similar tightening of bromopyruvate binding in the E-NADP+ (NADPH)-Mn2+ (Mg2+)-BP complexes. During alkylation, bromopyruvate interacts initially at the carboxylic acid substrate pocket of the active site, as indicated by the protective effect of substrates and the ability of this compound to form kinetically viable complexes with malic enzyme, particularly as a competitive inhibitor of pyruvate carboxylation with a Ki (90 muM) in the same order as its apparent Michaelis constant of 98 muM. Subsequent alkylation of a cysteinyl residue blocks the C-C bond cleavage step. The incorporation of radioactivity from [14C]bromopyruvate gives a half-site stoichiometry of two carboxyketomethyl residues per tetramer, indicating strong negative cooperativity between the four subunits of equal size, or alternatively the presence of structurally dissimilar active sites.

62] Mandelate racemase

Publisher Summary This chapter explains Mandelate racemase (EC 5.1.2.2) of Pseudomonas putida. It catalyzes the reversible interconversion of the D and L enantiomers of mandelate. The enzyme has been isolated in a high state of purity and has been shown to be a tetramer composed of identieal subunits, each with a molecular weight of 69,500. The enzyme acts without flavin or pyridine nucleotide cofactor, but has an absolute divalent metal ion requirement for activity. Mechanistic studies have indicated that the hydrogen alpha to the carboxylate is removed as a proton in the course of the enzyme-catalyzed racemization, generating a carbanion intermediate; the intermediate can be represented by the resonance structures. It followed that a basic group on the enzyme itself was participating in the generation of the carbanion intermediate by abstracting the a-hydrogen from mandelate. In an attempt to identify such a basic group on the enzyme, structural analogs of mandelate were sought that could also serve as alkylating agents. The chapter further explains the design of the affinity labeling reagent, synthesis of radioactivity labeled affinity label, determination of the stoichiometry of binding of the affinity label to the racemase, kinetic studies of the irreversible inhibition, effect of divalent metal ion on the affinity labeling process and attempts to identify the residue of the racemase alkylated by the affinity label.

Progesterone-binding components of chick oviduct. Biochemical characterization of purified oviduct progesterone receptor B subunit.

A number of physical and chemical properties of pure hen oviduct progesterone receptor B subunit have been determined. The molecule consists of a single polypeptide chain with a molecular weight of 115,000 g/mol as determined by gel filtration in the presence of 6 M guanidine hydrochloride and by gel electrophoresis in sodium dodecyl sulfate. The labeled subunit has retained the biologically important properties which it displayed in cruder preparations: it binds to nuclei (Kd = 1 X 10(-9) M) and chromatin (Kd = 1.5 X 10(-9) M) but does not bind to DNA. Reaction of the purified subunit with dansyl (5-dimethylaminonaphthalene-1-sulfonyl) chloride revealed a single NH2-terminal lysine. The amino acid composition has been determined and has been shown to be distinct from that of other steroid-binding proteins and consistent with the known properties of the molecule. In addition, no evidence for carbohydrate or phosphorylated amino acids was observed. The protein contains about 12% alpha helix as determined by circular dichroism. The ultraviolet spectrum of intact steroid receptor complexes revealed that the purified subunit had no pyridine nucleotide cofactor or nucleic acid, and that each receptor molecule contains a single hormone binding site. Electron microscopic analysis confirms the prolate-ellipsoid shape of the protein, with a long axis of 114 A. The purified protein isolated as described in a companion paper is shown here to have the characteristics of the crude receptor subunit B. Due to the apparent role in the hormone response, this protein has been named progestophilin B.