Germline transmission of exogenous genes in the chicken.

Difficulties associated with in vitro manipulation and culture of the early chicken embryo have restricted generation of transgenic chickens to approaches that use replication-competent retroviruses. The need to produce transgenic chickens in the absence of replicating virus prompted development of a new method of gene transfer into the chicken. Microinjection of the replication-defective reticuloendotheliosis virus (REV) vector ME111 beneath unincubated chicken embryo blastoderms results in infection of germline stem cells. This vector contains genetic information exogenous to the chicken genome, including both the herpes simplex virus type 1 thymidine kinase gene and the Tn5 neomycin phosphotransferase gene. About 8 percent of male birds hatched from injected embryos contained vector DNA in their semen. All four positive males tested passed vector sequences onto their progeny. Analysis of G1 offspring showed that gonads of G0 male birds were mosaic with respect to insertion of vector provirus. Thus, primordial germ cells present in the unincubated chicken embryo blastoderm are susceptible to infection by defective REV vectors.

Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

Two chimeric helper proviruses were derived from the provirus of the ecotropic Moloney murine leukemia virus by replacing the 5'long terminal repeat and adjacent proviral sequences with the mouse metallothionein I promoter. One of these chimeric proviruses was designed to express the gag-pol genes of the virus, whereas the other was designed to express only the env gene. When transfected into NIH 3T3 cells, these helper proviruses failed to generate competent virus but did express Zn2+-inducible trans-acting viral functions needed to assemble infectious vectors. One helper cell line (clone 32) supported vector assembly at levels comparable to those supported by the Psi-2 and PA317 cell lines transfected with the same vector. Defective proviruses which carry the neomycin phosphotransferase gene and which lack overlapping sequence homology with the 5' end of the chimeric helper proviruses could be transfected into the helper cell line without generation of replication-competent virus. Mass cultures of transfected helper cells produced titers of about 10(4) G418r CFU/ml, whereas individual clones produced titers between 0 and 2.6 X 10(4) CFU/ml. In contrast, defective proviruses which share homologous overlapping viral sequences with the 5' end of the chimeric helper proviruses readily generated infectious virus when transfected into the helper cell line. The deletion of multiple cis-acting functions from the helper provirus and elimination of sequence homology overlapping at the 5' ends of helper and vector proviruses both contribute to the increased genetic stability of this system.

3D simulations of hydrodynamic drag forces on two porous spheres moving along their centerline.

This paper numerically evaluates the hydrodynamic drag force exerted on two highly porous spheres moving steadily along their centerline through a quiescent Newtonian fluid over a Reynolds number ranging from 0.1 to 40. At creeping-flow limit, the drag forces exerted on both spheres were approximately identical. At higher Reynolds numbers the drag force on the leading sphere (sphere #1) was higher than the following sphere (sphere #2), revealing the shading effects produced by sphere #1 on sphere #2. At dimensionless diameter beta<2 (beta=d(f)/2k(0.5), d(f) and k are sphere diameter and interior permeability, respectively), the spheres can be regarded as "no-spheres" limit. At increasing beta for both spheres, the drag force on sphere #2 was increased because of the more difficult advective flow through its interior, and at the same time the drag was reduced owing to the stronger wake flow produced by the denser sphere #1. The competition between these two effects leads to complicated dependence of drag force on sphere #2 on beta value. These effects were minimal when beta became low.

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 beta-ketoacyl reductase or beta-ketoacyl dehydratase activity. The second-order rate constant of 0.2 mM-1 X s-1 for loss of synthetase activity is equal to the value for enoyl 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.

Duck liver malic enzyme: sequence of a tryptic peptide containing the cysteine residue labeled by the substrate analog bromopyruvate.

Malic enzyme of duck liver is alkylated by bromopyruvate with half-of-the-sites stoichiometry, and with accompanying loss of oxidative decarboxylase and enhancement of pyruvate reductase activities as was previously shown for the pigeon enzyme (Hsu, R.Y. (1982) Mol. Cell. Biochem. 43, 3-26). In the present work, the alkylated enzyme is shown to bind NADPH, but not L-malate in the presence of MnCl2, indicating impairment of the enzyme site for the substrate and/or divalent metal. The enzyme was differentially labeled by 3-bromo-1-[14C]-pyruvate and digested with TPCK-treated trypsin. Two peptides bearing the susceptible residue were purified by high-performance liquid chromatography and sequenced. Peptide II has the sequence of FMPIVYTPTVGLAXQQYGLAFR, corresponding to residues 86-107 (temporary numbering) of the duck enzyme; cysteine-99(x) is not detected, indicating that it is the target of modification by bromopyruvate. Peptide I is a truncated form of peptide II lacking five amino acid residues at the C-terminal. Cysteine-99 is conserved in malic enzymes from duck, rat, mouse, maize, human, Flaveria trinervia and Bacillus stearothermophilus.

Mechanism of pigeon liver malic enzyme modification of histidyl residues by ethoxyformic anhydride.

Incubation of malic enzyme (L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) with ethoxyformic anhydride caused the time-dependent loss of its ability to catalyze reactions requiring the nucleotide cofactor NADP+ or NADPH, such as the oxidative decarboxylase, the NADP+ - stimualted oxalacetate decarboxylase, the pyruvate reductase, and the pyruvate-medium proton exchange activities. Similar loss of oxidative decarboxylase and pyruvate reductase activities was affected by photo-oxidation in the presence of rose bengal. The inactivation of oxidative decarboxylase activity by ethoxyformic anhydride was accompanied by the reaction of greater than or equal to 2.3 histidyl residues per enzyme site and was strongly inhibited by NADP+. Ethoxyformylation also impaired the ability of malic enzyme to bind NADP+ or NADPH. These results support the involvement of histidyl residue(s) at the nucleotide binding site of malic enzyme.

Separation of active enzyme components from the fatty acid synthetase of chicken liver.

Fatty acid synthetase (EC 6.2.1.-) of chicken liver was dissociated into half-size subcomplexes and then separated into three protein fractions by the preparative disc-gel electrophoresis technique. The anodal protein (Fa) of a molecular weight of approx. 6000 contains the prosthetic group, 4'-phosphopantetheine. It binds acetyl group from acetyl-CoA and is identified as the acyl carrier protein component. The slower moving proteins (FI and FII) correspond to the subcomplexes resolved by the analytical method (Y.n, S.L. and Hsu, R.Y. (1972) J. Biol. Chem 247, 2689--2698). Both contain acetyl transacylase and palmityl-CoA deacylase activities, but only FI contains beta-ketoacyl reductase activity. Active acetyl transacylase and palmityl-CoA deacylase components were obrained by the sucrose density centrifugation technique in a broad 3 S protein band from the FI fraction, following dissociation at 4 degrees C for 12 days. Slight modification of the electrophoresis conditions yields a homogeneous 1.55 S beta-ketoacyl reductase component.

The pyruvate-proton exchange reaction of malic enzyme from pigeon liver.

Malic enzyme (L-malate:NADP+ oxidoreductase (decarboxylating) EC 1.1.1.40) catalyzes the incorporation of proton from medium water into pyruvate present either as the initial substrate or as the enzyme-bound product of malate decarboxylation. In the later reaction a single proton is incorporated into the methyl group of pyruvate. The pyruvate-medium proton exchange reaction requires Mg2+, NADPH and CO2-HCO3- as cofactors. The apparent Michaelis constants of pyruvate, NADPH and CO2-HCO3- are 4.8 mM, 2 microM and approx. 9 microM, respectively. The experimentally determined incorporation of 2.5 tritium atoms from tritiated water into pyruvate indicates that all three methyl protons of this compound are stereochemically equivalent in the exchange reaction. These results are consistent with the postulated kinetic mechanism for the malate reaction (Hsu, R.Y., Lardy, H.A. and Cleland, W.W. (1967) J. Biol. Chem. 242, 5315--5322), which predicts the formation of an enolpyruvate intermediate during the reaction. The rate of malic enzyme-catalyzed detritiation of beta-tritiated pyruvate is unaffected by modification of an essential protein thiol group with 5,5'-dithiobis(2-nitrobenzoic acid) or KCN. Moreover, the native- and thiol-modified enzymes also catalyze the detritiation of beta-tritiated bromopyruvate at slower rates.

The presence of essential arginine residues at the NADPH-binding sites of beta-ketoacyl reductase and enoyl reductase domains of the multifunctional fatty acid synthetase of chicken liver.

Treatment of chicken liver fatty acid synthetase with the arginine-specific reagent phenylglyoxal resulted in the pseudo-first-order loss of synthetase, beta-ketoacyl reductase and enoyl reductase activities. The sum of the second-order rate constants for the two reductase reactions equalled that for the synthetase reaction, suggesting that inactivation of either reductase was responsible for the loss of fatty acid synthetase activity. Double-log plots of pseudo-first-order rate constant versus reagent concentration yielded straight lines with slopes of unity for all three activities tested, suggesting the reaction of one reagent molecule in the inactivation process. In parallel experiments, complete inactivation of synthetase activity was accompanied by the incorporation of 4.5 [14C]phenylglyoxal, and the loss of 2.3 arginine residues per subunit. Reaction of essential sulfhydryl groups was not involved, since inactivation by phenylglyoxal was unaffected by reversible protection of these groups with 5,5'-dithiobis(2-nitrobenzoic acid). Inactivation of all three activities by phenylglyoxal was prevented by saturating amounts of the coenzyme NADPH, or its analogs 2',5'-ADP and 2'-AMP, but not by the corresponding nucleotides containing only the 5'-phosphate. Conversely, the ability of this enzyme to bind NADPH was abolished upon inactivation. These results are consistent with the presence of an essential arginine residue at the binding site for the 2'-phosphate group of NADPH at each of the two reductase domains of the multifunctional fatty acid synthetase subunit.

Affinity labeling of chicken liver fatty acid synthase with chloroacetyl-CoA and bromopyruvate.

Fatty acid synthase of chicken liver is inactivated rapidly and irreversibly by incubation with chloroacetyl-CoA or with bromopyruvate. Inactivation by both reagents follows saturation kinetics, indicating the formation of an E ... I complex (dissociation constants of 0.36 microM for chloroacetyl-CoA and 31 microM for bromopyruvate) prior to alkylation. The limiting rate constants are 0.15 s-1 for bromopyruvate and 0.041 s-1 for chloroacetyl-CoA. Inactivation by both reagents is protected by NADPH and 200 mM KCl, and by saturating amounts of thioester substrates which reduced the limiting rate constants 6.5-30-fold. Active-site-directed reaction of chloroacetyl-CoA is supported by the ability of this compound to form a kinetically viable complex with the enzyme as competitive inhibitor of acetyl-CoA. Chloroacetyl-CoA interacts initially at the CoA binding pocket, since the nucleotide afforded competitive protection of inactivation and caused a large decrease in its affinity. Subsequently, the phosphopantetheine prosthetic group is alkylated. Evidence is presented to show that bromopyruvate competes with chloroacetyl-CoA for the same target site.

Reduction of alpha-oxo carboxylic acids by pigeon liver 'malic' enzyme.

1. Pigeon liver ;malic' enzyme [l-malate-NADP(+) oxidoreductase (decarboxylating); EC 1.1.1.40] was shown to catalyse the reductase reaction: [Formula: see text] l-Malate was identified as the reaction product, and was formed in stoicheiometric amount. 2. In addition to oxaloacetate and pyruvate, a number of other alpha-oxo carboxylic acids were also reduced.

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.

Pigeon liver malic enzyme: involvement of an arginyl residue at the binding site for malate and its analogs.

Treatment of malic enzyme with arginine-specific reagents phenylglyoxal or 2,3-butanedione results in pseudo-first-order loss of oxidative decarboxylase activity. In-activation by phenylglyoxal is completely prevented by saturating concentrations of NADP+, Mn2+, and substrate analog hydroxymalonate. Double log plots of pseudo-first-order rate constant versus concentration yield straight lines with identical slopes of unity for both reagents, suggesting that reaction of one molecule of reagent per active site is associated with activity loss. In parallel experiments, complete inactivation is accompanied by the incorporation of four [14C]phenylglyoxal molecules, and the loss of two arginyl residues per enzyme subunit, as determined by the colorimetric method of Yamasaki et al. (R. B. Yamasaki, D. A. Shimer, and R. E. Feeney (1981) Anal. Biochem. 14, 220-226). These results confirm a 2:1 ratio for the reaction between phenylglyoxal and arginine (K. Takahashi (1968) J. Biol. Chem. 243, 6171-6179) and yield a stoichiometry of two arginine residues reacted per subunit for complete inactivation, of which one is essential for enzyme activity as determined by the statistical method of Tsou (C. L. Tsou (1962) Acta Biochim. Biophys. Sinica 2, 203-211) and the Ray and Koshland analysis (W. J. Ray and D. E. Koshland (1961) J. Biol. Chem. 236, 1973-1979). Amino acid analysis of butanedione-modified enzyme also shows loss of arginyl residues, without significant decrease in other amino acids. Modification by phenylglyoxal does not significantly affect the affinity of this enzyme for NADPH. Binding of L-malate and its dicarboxylic acid analogs oxalate and tartronate is abolished upon modification, as is binding of the monocarboxylic acid alpha-hydroxybutyrate. The latter result indicates binding of the C-1 carboxyl group of the substrate to an arginyl residue on the enzyme.

Equilibrium substrate binding studies of the malic enzyme of pigeon liver. Equivalence of nucleotide sites and anticooperativity associated with the binding of L-malate to the enzyme-manganese(II)-reduced nicotinamide adenine dinucleotide phosphate ternary complex.

Malic enzyme (ME) from pigeon liver is a tetrameric protein containing apparently identical subunits. In the present study, equilibrium dialysis and fluorescence titration techniques are employed to determine the binding parameters of nucleotide cofactors, malate, and the inhibitor oxalate. ME binds NADP+ or NADPH at four independent and equivalent sites with dissociation constants of 1.33 microM (pH 7.5, 4 degrees C) and 0.29 microM (pH 7.0, 5 degrees C), respectively, showing "all-of-the-sites" reactivity. The affinity of both nucleotides decreases with increasing temperature, yielding delta Hdissociation values of 11.4 kcal/mol for E-NADP+ and 8.9 kcal/mol for E-NADPH, thus implicating the involvement of polar forces in the binding process. The affinity of NADP+ is independent of pH between 6.1 and 8.4 whereas that of NADPH is highly pH dependent and decreases approximately 63-fold from pH 6.0 to pH 8.0. The pH profile suggests the participation of a protonated enzyme group(s) (pK = 7.2-7.5) in NADPH binding, probably a histidine residue. The affinity of NADP+ is enhanced ca. twofold by pyruvate, in the presence of Mn2+ (50-100 microM) saturating only two "tight" metal sites [Hsu, R. Y., Mildvan, A. S., Chang, G. G., & Fung, C. H. (1976) J. Biol. Chem. 251, 6574]. Binding of Mn2+ at weak metal sites (KD congruent to 0.9 mM) prevents this change. Malate binds free ME or binary E-Mn2+ and E-NADP+ (H) complexes weakly with dissociation constants of greater than or equal to 2 mM. The affinity is significantly increased by Mn2+ and NADPH in the ternary E-Mn2+-NADPH complex, yielding two "tight" (KD = 22-30 microM) and two "weak" (KD = 250-400 microM) malate sites per enzyme tetramer as the result of either preexisting nonidentity or negative cooperativity between intitially identical sites. The transition-state inhibitor oxalate binds ME tightly (KD = 65 microM) at the two tight malate sites, showing "half-of-the-sites" stoichiometry. The binding parameters are unaffected by Mn2+, whereas the affinity of this inhibitor is enhanced 3.5-fold by saturation with NADPH. Further evidence for the half-of-the-sites reactivity of the affinity label bromopyruvate [Pry, T. A., & Hsu, R. Y. (1978) Biochemistry 17, 4024] is obtained by sequential modification of the four putatively identical SH groups of ME with bromopyruvate, 5,5'-dithiobis(2-nitro-benzoic acid), and K14CN. The modified enzyme has a structure of E4(S-pyr)2(S-14CN)2 and is "inactive" in the reaction with malate. In contrast, the E(S-14CN)4 derivative prepared in the absence of bromopyruvate is completely active. The oxidative decarboxylase reaction is inhibited by high concentrations (greater than or equal to 0.3 mM) of malate in the presence of tightly bound Mn2+. Direct binding studies show a parallel increase in the affinity of NADPH, confirming our previous notion [Reynolds, C. H., Hsu, R. Y., Matthews, B., Pry, T. A., & Daibits the rate-limiting NADPH release step.

Studies on the reactivity of the essential sulfhydryl groups as a conformational probe for the fatty acid synthetase of chicken liver. Inactivation by 5,5'-dithiobis-(2-nitrobenzoic acid) and intersubunit cross-linking of the inactivated enzyme.

Fatty acid synthetase of chicken liver is rapidly and reversibly inactivated by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) at a rate (k2 = 132 mM-1 S-1 in 3 mM EDTA, 1% (v/v) glycerol, pH 7.0, at 25 degrees C) up to 2200 times higher than the reaction of this reagent with simple thiol compounds. The inactivation is caused by the reaction of the phosphopantetheine SH group, since it is protected competitively by either acetyl- or malonyl-CoA, and since the inactivated enzyme is unreactive with the phosphopantetheine label chloroacetyl-CoA but reactive with the cysteine reagent 1,3-dibromopropanone. Moreover, chloroacetyl-CoA prevents the modification of the rapidly reacting essential SH group by DTNB. The number of SH groups involved in inactivation was determined by correlating activity loss with the extent of reaction and by stopped-flow analysis of substrate (or chloroacetyl-CoA) protection. Values between 0.91 and 1.15 SH groups/dimer were obtained, indicating the presence of substoichiometric amounts of the prosthetic group in the fatty acid synthetase preparations used in this study. Inactivation of the synthetase by DTNB is strongly inhibited by increasing salt concentration and protected noncompetitively by NADP+ and NADPH. Treatment of the enzyme inactivated at low salt by salt, NADP+, or NADPH also effectively reduced cross-linking between enzyme subunits. The parallel effects of these treatments on the reaction with DTNB and subsequent dimerization are consistent with a minimum model of two discreet conformation states for fatty acid synthetase. In the low salt conformer, the phosphopantetheine and cysteine SH groups are juxtaposed, and the DTNB reaction (k2 approximately 132 mM-1 S-1) and dimerization are both facilitated. Transition to the high salt conformer by the above treatments is accompanied by an approximately 20-fold reduction of reactivity with DTNB (k2 = 6.8 mM-1 S-1) and reduced dimerization, due to spatial separation of the SH groups. During palmitate synthesis, the enzyme may oscillate between these conformation states to permit the reaction of intermediates at different active sites. Results obtained by studies on the effect of pH on DTNB inactivation implicate a pK of 5.9-6.1 for the essential SH group independent of salt concentration. This value is 1.5-1.8 pH units lower than the pK of 7.6-7.7 for CoA and may explain the 23-fold increase of the rate constant from a value of 0.3 mM-1 S-1 for CoA to that of the high salt conformer.

Induction of fatty acid synthetase synthesis in differentiating 3T3-L1 preadipocytes.

3T3-L1 preadipocytes, cloned from 3T3 mouse embryo fibroblasts, differentiate in monolayer culture into cells with morphological and biochemical characteristics of adipocytes. Deposition of cytoplasmic triglyceride is associated with an increased lipogenic rate and a coordinate rise in the activities of many lipogenic enzymes (Mackall, J.C., Student, A.K., Polakis, S.E., and Lane, M.D. (1976) J. Biol. Chem. 251, 6462-6464). During differentiation induced by a 48-h treatment of postconfluent cells with methylisobutylxanthine, dexamethasone, and insulin, fatty acid synthetase activity increased to a level 19.5-fold higher than that of undifferentiated 3T3-L1 cells or nondifferentiating 3T3-C2 cells. The rate of [3H]leucine incorporation into immunoadsorbable fatty acid synthetase rose to a maximum and then declined to a new level 12.5-fold higher in differentiated than in undifferentiated 3T3-L1 cells. The kinetics of the changing [3H]leucine incorporation rate was reflected in the kinetics of the rise in fatty acid synthetase activity. The rate of degradation of fatty acid synthetase, determined by pulse-chase experiments, was unaffected by differentiation, the t1/2 remaining constant at 1.4 days. It is concluded that the higher level of fatty acid synthetase activity in differentiated 3T3-L1 cells can be attributed entirely to an increased rate of enzyme synthesis. The rate of total cellular protein synthesis also increases early early in differentiation, lending support to a model in which the synthesis of a large number of "differentiated proteins" is coordinately induced.

Hybridization studies of chicken liver fatty acid synthetase. Evidence for the participation in palmitate synthesis of cysteine and phosphopantetheine sulfhydryl groups on adjacent subunits.

Enzymatically inactive variants of chicken liver fatty acid synthetase have been prepared by specific chemical modification of the active cysteine SH group with iodoacetamide, and the phosphopantetheine SH group with chloroacetyl-CoA. Hybridization of each of these variants with the unmodified enzyme yielded (modified)-(unmodified) hybrid dimers which possessed 50% synthetase activity. A 50% active (iodoacetamide-modified)-(chloroacetyl-CoA-modified) hybrid dimer was also demonstrated by recombination of these variants with each other. These results indicate that the two functional sites on the synthetase are independently active, and that each is comprised of a cysteine SH group from one subunit and a complementary phosphopantetheine SH group from the other subunit as depicted by the head-to-tail arrangement proposed by Wakil and co-workers (Wakil, S. J., Stoops, J. K., and Joshi, V.C.

Essential sulfhydryl group of malic enzyme from Escherichia coli.

The activity of malic enzyme from Escherichia coli was unaffected by the monovalent cations Na+ or Li+ at 10 mM. At 100 mM, Li+ or Na+ inhibited the enzyme activity by 88% and 83%, respectively. However, the enzyme activity was stimulated by 40-80-fold with 10 mM K+, Rb+, Cs+, or NH4+. Less stimulation was observed with 100 mM of these stimulating cations. The stimulatory effect was lost after the enzyme was dialyzed against Tris-Cl buffer, but was regained after incubating the dialyzed enzyme with dithiothreitol. The regenerated enzyme was inactivated by 5,5'-dithiobis(2-nitrobenzoic acid). The resulting inactive thionitrobenzoyl enzyme could be regenerated to the active thiol-enzyme by dithiothreitol or converted to the inactive thiocyanoylated enzyme by KCN. The thiocyanoylated enzyme was insensitive to K+ stimulation, which suggested the essentiality of the sulfhydryl groups of the E. coli malic enzyme.

Mechanism of malic enzyme from pigeon liver. Magnetic resonance and kinetic studies of the role of Mn2+.

As determined by EPR, malic enzyme from pigeon liver binds Mn2+ with a half-site stoichiometry of two tight binding sites (KD=6 to 10 mum) per enzyme tetramer and at two to four weak binding sites (KD=0.43 to 1.34 mM). The activation of malic enzyme by Mn2+ at high levels of L-malate shows biphasic kinetics yielding two activator constants for Mn2+. The dissociation constants of Mn2+ for both classes of sites are of the same order as the kinetically determined activator constants of Mn2+, indicating active site binding at both classes of binding sites. The binding of Mn2+ to the tight sites enhances the paramagnetic effect of Mn2+ on 1/T1 of water protons by a factor (epsilon) of 17, while binding at the weak sites yields a smaller epsilon of 11. The coenzymes TPN and TPNH have no effects on epsilon, while the carboxylic acid substrates L-malate and pyruvate and the inhibitors D-malate and oxalate significantly decrease epsilon. TPNH causes a 38-fold tightening of binding of the substrate L-malate to the enzyme-Mn2+ complex, consistent with the previously described highly ordered kinetic scheme, but only a 2-fold tightening of binding of the competitive inhibitor D-malate. The dissociation constant of L-malate from the quaternary E-Mn2+-TPNH-L-malate complex (32 muM) agrees with the Km of L-malate (25 muM), indicating active site binding. The dissociation constants of pyruvate from the ternary E-Mn2+-pyruvate complex (12 mM) and from the quaternary E-Mn2+-TPN-pyruvate complex (20 mM) are similar to the Km of pyruvate (5 mM), also indicating active site binding and a less highly ordered kinetic scheme for the reactions of pyruvate than for those of L-malate. Analysis of the frequency dependence of 1/T1 of water protons indicates that two fast exchanging water ligands remain coordinated to Mn2+ in the binary E-Mn2+ complex. The binding of the substrates L-malate and pyruvate and of the transition state analog oxalate to the E-Mn2+ complex decrease the number of fast exchanging water ligands on Mn2+ by approximately 1, but the binding of D-malate has no significant effect on this parameter, indicating the occlusion or replacement of a water ligand of the enzyme-bound Mn2+ by a properly oriented substituent on C-2 of the substrate. Occlusion rather than replacement of a water ligand by pyruvate is established by studies of 1/T1 of 13COO- and 13CO-enriched pyruvate which indicate second sphere Mn2+ to pyruvate distances of 4.6 A (COO-) and 4.8 A (CO) in the ternary enzyme-Mn2+-pyruvate complex. Formation of the quaternary complex with TPN increases these distances by 0.8 A, indicating the participation of a second sphere enzyme-Mn2+-(H2O)-pyruvate complex in catalysis. Thus, malic enzyme, like five other enzymes which utilize metals to polarize carbonyl groups, forms a second sphere complex with its substrate.