Purification and molecular weight determination of glucose-6-phosphate dehydrogenase and malic enzyme from mouse and Drosophila.

A facile two-step procedure was employed for simultaneous purification of glucose-6-phosphate dehydrogenase and malic enzyme from mouse (strain $\text{DBA}\text{2J}$) and Drosophila melanogaster. This involved the use of an 8-(6-aminohexyl)-amino-2′,5′-ADP-Sepharsoe affinity column chromatography followed by DEAE-Sephadex chromatography. The native and subunit molecular weights of these two homogeneous enzymes were determined by gel-filtration chromatography and SDS-polyacrylamide gel electrophoresis. From this study, it was concluded that the two enzymes are tetrameric and have native molecular weights between 200,000 and 280,000 in both species.     

Investigation into mechanism of orotidine 5′-monophosphate decarboxylase enzyme by MM-PBSA/MM-GBSA and molecular docking

In this work, we studied the binding affinity of orotidine 5′-monophosphate (OMP) and 6-hydroxy-UMP (BMP) for Saccharomyces cerevisiae orotidine 5′-monophosphate decarboxylase (OMPDC) enzyme by using Molecular Mechanics-Poisson–Boltzmann Surface Area (MM-PBSA) and the Molecular Mechanics-Generalised Born Surface Area (MM-GBSA) calculations. In all simulations, Asp91, which is an important residue in the enzyme active site, was considered in both anionic (present in the native form of the enzyme) and neutral states. A series of 10-ns molecular dynamics simulations were performed for the four OMPDC–ligand complexes, two ligand-free enzymes and two free ligands, followed by MM-PBSA/MM-GBSA calculations on the collected snapshots, and molecular docking calculations using the free enzymes and ligands. The results of MM-PBSA/MM-GBSA calculations indicate that all of the OMPDC–ligand complexes form favourable systems in water, which is in agreement with corresponding experimental data. The results of the MM-PBSA and molecular docking methods also showed that OMPDC–BMP complexes, transition state analogue and inhibitor of the OMPDC enzyme have the highest binding affinities. The fact that in the native anionic state BMP shows a higher binding affinity compared with the substrate suggests the contribution of a transition state stabilisation mechanism in the debatable catalytic mechanism of the OMPDC enzyme.     

Evidence that the conformational stability of 'aged' organophosphate-inhibited cholinesterase is altered

In order to determine whether a structural modification at the active center of cholinesterase may alter the conformational stability of the enzyme we compared the urea-induced unfolding of the tetrameric form of non-inhibited and irreversibly inhibited human plasma cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8). We studied enzyme inhibited by methanesulfonyl fluoride, diisopropylfluorophosphonate (DFP) and racemic soman. DFP- and soman-inhibited cholinesterases are converted spontaneously into non-reactivable forms called 'aged' enzymes through a process involving dealkylation of the bound organophosphate residue. The unfolding was followed by transverse urea-gradient polyacrylamide electrophoresis at various temperatures ranging from 0 to 60 degrees C. Unfolding of cholinesterase appears to be a complex process. The denaturation patterns showed that partially unfolded states are thermodynamically unstable, but that several intermediates are involved; the lifetime of these depends on the temperature at which electrophoreses are carried out. Cholinesterase inhibited by methanesulfonyl fluoride behaved like the non-inhibited enzyme. On the other hand, small but significant differences in stability between non-inhibited and aged enzymes were observed. Whatever the temperature, the urea concentration at the mid-point of transition was always greater for aged enzyme than for the non-inhibited enzyme. In addition, aged enzymes showed more complex denaturation patterns at the lower temperatures (under 20 degrees C). These findings suggest that the overall stability of aged-cholinesterases is slightly increased as compared with the stability of non-inhibited or methanesulfonyl fluoride-inhibited enzymes. The denaturation pattern obtained at 0 degree C for soman-inhibited cholinesterase under non-aging conditions (inhibition at 0 degree C, pH 10.7) was similar to that of non-inhibited enzyme at this temperature, although splitting in two of the denaturation curve over the transition zone reflects the heterogeneity of soman-inhibited enzyme. The slight difference in denaturation behavior between these species may be due to stereoisomerism in soman. The differences in electrophoretic behavior and apparent stability observed between non-inhibited and aged enzymes were interpreted as the result of a conformational change induced by the dealkylation reaction of enzyme-inhibitor conjugates.     

Structural and functional investigations of cholinesterases by means of affinity electrophoresis

1. After a brief survey of the basic affinity electrophoresis concepts, the usual ways for preparing affinity electrophoresis ligands are examined. 2. Then results obtained on cholinesterases are reviewed. This section includes (a) structural and functional investigations on anionic sites, i.e., study of ligand-induced conformational change, organophosphate-induced "aging," genetic variants, and active-site topology; and (b) characterization of cholinesterase conjugates (hybrid proteins) and glycoinositol phospholipid-anchored cholinesterases. 3. The future prospects of affinity electrophoresis, e.g., investigations on the esteratic site and exploration of the carbohydrate moiety, are emphasized in the concluding section.     

Further studies on aspartate aminotransferase of thermophilic methanogens by analysis of general properties, bound cofactors, and subunit structures.

Aspartate aminotransferase (AspAT) [EC 2.6.1.1] of thermophilic methanogen was further characterized with the enzyme from Methanobacterium thermoautotrophicum strain FTF-INRA as well as M. thermoformicicum strain SF-4. AspAT of strain FTF-INRA was similar in the amino donor specificity to the enzyme of M. thermoformicicum strain SF-4, in that it was active on L-cysteine and L-cysteine sulfinate in addition to L-glutamate and L-aspartate. The enzymes gave similar absorption spectra having maxima at around 326 and 415 nm with no pH-dependent shift but were found to contain 1 mol of tightly bound pyridoxal 5'-phosphate (PLP) per subunit. Reconstitution of each apoenzyme with added PLP resulted in partial recovery of the original enzymatic activity, suggesting a significant conformational change of the active site region upon removal of the cofactor. Polyacrylamide gel electrophoresis (PAGE) and gel filtration analyses revealed a tetrameric structure (180 kDa) of identical subunits with a molecular mass of 43 kDa for each of these enzymes. Electric current was found to affect the interaction or affinity of each subunit, promoting dissociation of the native enzyme into the monomeric form. Alkaline treatment was effective only for dissociation of the enzyme from strain SF-4. They were distinguishable by the more rapid reassociation of the monomer to the native aggregated form in the enzyme of strain FTF-INRA.     

Evidence that the conformational stability of ‘aged’ organophosphate-inhibited cholinesterase is altered

In order to determine whether a structural modification at the active center of cholinesterase may alter the conformational stability of the enzyme we compared the urea-induced unfolding of the tetrameric form of non-inhibited and irreversibly inhibited human plasma cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8). We studied enzyme inhibited by methanesulfonyl fluoride, diisopropylfluorophosphonate (DFP) and racemic soman. DFP- and soman-inhibited cholinesterases are converted spontaneously into non-reactivable forms called ‘aged’ enzymes through a process involving dealkylation of the bound organophosphate residue. The unfolding was followed by transverse urea-gradient polyacrylamide electrophoresis at various temperatures ranging from 0 to 60°C. Unfolding of cholinesterase appears to be a complex process. The denaturation patterns showed that partially unfolded states are thermodynamically unstable, but that several intermediates are involved; the lifetime of these depends on the temperature at which electrophoreses are carried out. Cholinesterase inhibited by methanesulfonyl fluoride behaved like the non-inhibited enzyme. On the other hand, small but significant differences in stability between non-inhibited and aged enzymes were observed. Whatever the temperature, the urea concentration at the mid-point of transition was always greater for aged enzyme than for the non-inhibited enzyme. In addition, aged enzymes showed more complex denaturation patterns at the lower temperatures (under 20°C). These findings suggest that the overall stability of aged-cholinesterases is slightly increased as compared with the stability of non-inhibited or methanesulfonyl fluoride-inhibited enzymes. The denaturation pattern obtained at 0°C for soman-inhibited cholinesterase under non-aging conditions (inhibition at 0°C, pH 10.7) was similar to that of non-inhibited enzyme at this temperature, although splitting in two of the denaturation curve over the transition zone reflects the heterogeneity of soman-inhibited enzyme. The slight difference in denaturation behavior between these species may be due to stereoisomerism in soman. The differences in electrophoretic behavior and apparent stability observed between non-inhibited and aged enzymes were interpreted as the result of a conformational change induced by the dealkylation reaction of enzyme-inhibitor conjugates.     

Possibilitiés d'étude des variantes de la cholinestérase plasmatique humaine par électrophorèse d'affinitéPossibilities of study of the human plasma cholinesterase variants by affinity electrophoresis

Affinity electrophoresis has been applied to the study of the multiple molecular forms of three human plasma cholinesterase phenotypes (usual enzyme U, atypical enzyme A and intermediate UA). Electrophoreses were carried out in polyacrylamide gels containing a water-soluble macromolecular derivative of m-amino-(substituted)-phenyltrimethylammonium immobilized within the gel network.Apparent dissociation constants (K_{D app}) were estimated from the mobilities of the enzymes versus ligand concentration.The ratio of K_{D app} values of the molecular forms of phenotypes A and U which is approximately 2 is consistent with the hypothesis that the anionic site is altered in atypical enzyme.     

Possibilitiés d'étude des variantes de la cholinestérase plasmatique humaine par électrophorèse d'affinitéPossibilities of study of the human plasma cholinesterase variants by affinity electrophoresis

Affinity electrophoresis has been applied to the study of the multiple molecular forms of three human plasma cholinesterase phenotypes (usual enzyme U, atypical enzyme A and intermediate UA). Electrophoreses were carried out in polyacrylamide gels containing a water-soluble macromolecular derivative of m-amino-(substituted)-phenyltrimethylammonium immobilized within the gel network.Apparent dissociation constants (K_{D app}) were estimated from the mobilities of the enzymes versus ligand concentration.The ratio of K_{D app} values of the molecular forms of phenotypes A and U which is approximately 2 is consistent with the hypothesis that the anionic site is altered in atypical enzyme.     

Stabilization of human prostate acid phosphatase by cross-linking with diimidoesters

1. Modification of dimeric human prostate acid phosphatase (EC 3.1.3.2) by diimidoesters leads to the formation of water-soluble preparations of high enzymatic activity, resistant to denaturing agents. 2. Monomeric, dimeric, trimeric and tetrameric species were found in SDS-polyacrylamide gel electrophoresis of the phosphatase cross-linked with dimethyl-suberimidate, and dimeric, trimeric and tetrameric enzymatically active species on thin-layer Sephadex 200 gel filtration. This molecular pattern evidenced formation of the inter-subunit covalent linkages. All molecular forms are immunoreactive against the polyclonal rabbit anti-phosphatase antibodies. 3. The catalytic properties of the modified phosphatase are almost the same as those of the native enzyme. Differences in the optical properties between the modified and the native enzymes point to slight conformational transitions in the modified enzyme.     

Evidence of the presence of a latent active site in the large subunit of rat kidney gamma-glutamyl transpeptidase.

γ-Glutamyl transpeptidase (EC 2.3.2.2) of rat kidney is composed of two nonidentical polypeptide chains, the small and large subunits. The active site of this enzyme has previously been shown to be located in the small subunit [Inoue, M., Horiuchi, S. &; Morino, Y. (1977) Eur, J. Biochem. $\text{73}$, 335–342; Tate, S. S. &; Meister, A. (1977) Proc. Natl. Acad. Sci. U.S.A. $\text{74}$, 931–935] The denaturation of this oligomeric enzyme in 6 M urea, followed by chromatography on a Sephadex G-150, resulted in the separation of the large and small subunits. The removal of urea gave rise to an enzymatically active preparation from the denatured large subunit. Under several renaturation conditions, the small subunit polypeptide chain did not exhibit the enzymatic activity. Upon incubation with 6-diazo-5-oxo-L-[1,2,3,4,5-¹⁴C]norleucine, an affinity label for γ-glutamyl transpeptidase, the renatured preparation of the large subunit was covalently labeled with the affinity label with concomitant loss of the enzymatic activity. When the native enzyme was inactivated by the ¹⁴C-affinity label, radioactivity was selectively incorporated into the small subunit. These findings indicate that the isolated large subunit possesses an active site which is masked in the native state of the enzyme.     

Chemical modification of mouse β-glucuronidase implicates lysyl,carboxyl and tyrosyl residues as catalytically essential and causes a reversible dissociation of the subunits

Maleic anhydride modification of tetrameric mouse β-glucuronidase, followed by a 70° incubation, dissociated the tetramer into inactive monomers. Deblocking of this derivative allowed 80% regeneration of activity and tetrameric structure. The enzyme was inactivated by reaction with N-ethyl-S-phenylisoxazolium-3′-sulfonate, tetranitromethane and succinic anhydride, but not when a competitive inhibitor was added to enzyme prior to modification. These data suggest that the active site residues of mouse β-glucuronidase include carboxyl, tyrosyl and lysyl residues. In comparison, it has been reported that rat β-glucuronidase is inactivated by chemical modification of carboxyl, tyrosyl and $\text{histidyl}$ residues.     

Investigation of the symmetry of oligomeric enzymes with bifunctional reagents.

The symmetry of proteins composed of identical polypeptide chains has been investigated by means of cross-linking with bifunctional reagents and subsequent sodium dodecylsulfate-polyacrylamide gel electrophoresis. The majority of the investigations were performed with diimidates of different chain lengths (C3-C12), which react exclusively with amino groups. Aldolase, catalase, fumarase, pyruvate kinase, tetrameric proteins with identical polypeptide chains, reveal a D2 symmetry, i.e. they appear to be composed of two pairs of polypeptide chains. The validity of this conclusion is demonstrated with lactate dehydrogenase. This enzyme, shown by X-ray analysis to have a D2 symmetry, yields after cross-linking and subsequent polyacrylamide electrophoresis the band pattern expected for a protein with this quaternary structure and similar to the pattern obtained with the above enzymes. 2. The influence of the experimental conditions on the cross-linking reaction has been investigated. The selectivity of the bifunctional reagent for the different contact domains within the quaternary structure of a protein depends on the reaction time, the chain length and on the concentration of the reagent. In general the D2 symmetry becomes more obvious with increasing chain length and with increasing concentration of the diimidate. Diethylpyrocarbonate showed very little selectivity.     

Subunit structures of AMP deaminase isozymes in rat

AMP deaminases A and B have been purified to apparent homogeneity from rat muscle and liver, respectively. The molecular weights of 286,000 and 351,000 were obtained for the native muscle and liver enzymes, respectively, by sedimentation equilibrium studies. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the muscle preparation exhibited a single polypeptide band with a molecular weight of 72,000; the liver preparation, a molecular weight of 85,000. The data indicate that each enzyme has a tetrameric structure.     

Biosynthesis of 5-methylaminomethyl-2-thiouridylate. I. Isolation of a new tRNA-methylase specific for 5-methylaminomethyl-2-thiouridylate

A new enzyme, which catalyzes the transfer of a methyl group to tRNA to form 5-methylaminomethyl-2-thiouridylate, was isolated from $\text{E.}$$\text{coli}$ by a procedure including affinity chromatography. The purified enzyme was nearly homogeneous upon disc electrophoresis. Using methyl-deficient tRNA^Glu of $\text{E.}$$\text{coli}$ as substrate, the 5-methylaminomethyl-2-thiouridylate residue synthesized was mostly found in the anticodon loop, showing a coincidence of the modification site $\text{in}$$\text{vitro}$ with that $\text{in}$$\text{vivo}$.     

Crystal structures of Salmonella typhimurium biodegradative threonine deaminase and its complex with CMP provide structural insights into ligand-induced oligomerization and enzyme activation

Two different pyridoxal 5'-phosphate-containing l-threonine deaminases (EC 4.3.1.19), biosynthetic and biodegradative, which catalyze the deamination of l-threonine to alpha-ketobutyrate, are present in Escherichia coli and Salmonella typhimurium. Biodegradative threonine deaminase (TdcB) catalyzes the first reaction in the anaerobic breakdown of l-threonine to propionate. TdcB, unlike the biosynthetic threonine deaminase, is insensitive to l-isoleucine and is activated by AMP. In the present study, TdcB from S. typhimurium was cloned and overexpressed in E. coli. In the presence of AMP or CMP, the recombinant enzyme was converted to the tetrameric form accompanied by significant enzyme activation. To provide insights into ligand-mediated oligomerization and enzyme activation, crystal structures of S. typhimurium TdcB and its complex with CMP were determined. In the native structure, TdcB is in a dimeric form, whereas in the TdcB.CMP complex, it exists in a tetrameric form with 222 symmetry and appears as a dimer of dimers. Tetrameric TdcB binds to four molecules of CMP, two at each of the dimer interfaces. Comparison of the dimer structure in the ligand (CMP)-free and -bound forms suggests that the changes induced by ligand binding at the dimer interface are essential for tetramerization. The differences observed in the tertiary and quaternary structures of TdcB in the absence and presence of CMP appear to account for enzyme activation and increased binding affinity for l-threonine. Comparison of TdcB with related pyridoxal 5'-phosphate-dependent enzymes points to structural and mechanistic similarities.     

Purification and partial characterization of the soluble low Km 5'-nucleotidase from human seminal plasma

Soluble low Km 5'-nucleotidase from human seminal plasma has been purified to homogeneity by one affinity and two gel-filtration chromatographic steps. The pure enzyme had a specific activity of 2000 nmol min-1 mg-1. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified low Km 5'-nucleotidase revealed a single polypeptide band of 40 +/- 7 kDa and a tetrameric structure of 160 +/- 10 kDa has been proposed for the native enzyme. The kinetic properties of low Km 5'-nucleotidase have been determined and rather unique characteristics have been found for this soluble low Km 5'-nucleotidase: the substrate efficiency was slightly higher for IMP with an optimum pH at 7.5; the enzyme showed an absolute dependence on Mg2+ ions. Ca2+ could replace Mg2+ ions for activity while other divalent cations could not substitute for Mg2+; the enzymes were equally activated by ATP and ADP up to 0.1 mM concentrations. At higher concentrations up to 1 mM, ADP was still an activator while ATP caused a gradual decrease of activation to the native activity. This effect could not be related to the Mg-ATP = complexes since the enzymic preparation Mg(2+)-free still showed the same biphasic pattern of activation.     

Glutathione S-transferases from the New Zealand grass grub,Costelytra zealandica Their isolation and characterization and the effect on their activity of endogenous factors

Isolation of glutathione $\text{S-}\text{transferase}$ from the New Zealand grass grub, is complicated by the marked loss of activity from crude homogenates. This loss may be due to proteolysis or to modification by endogenous chemicals. The effect may be minimized by immediate fractionation with ammonium sulphate and by inclusion of 5mM glutathione in homogenates.Two enzymes species, isoelectric at pH 8.7 and 5.9 respectively, could be isolated by ammonium sulphate fractionation, affinity chromatography, anion exchange chromatography and chromatography on hydroxyl apatite. They had different substrate specificities and had differing subunit structure. The pI 8.7 enzyme appeared to be a homodimer of subunits of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 23,700 and the pI 5.9 enzyme one of subunit $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 22,500.A third major enzyme species, isoelectric at pH 4.3 differed from the other two enzymes in having low affinity for the affinity matrix. This preparation was heterogeneous. The enzymically active species in this preparation had the same molecular weight as that of the pI 8.7 enzyme, had a very similar substrate specificity to the basic enzyme species and was characterized by kinetic parameters almost identical to those of the pI 8.7 enzyme.     

A model for the allosteric regulation of pH-sensitive enzymes.

1. In an enzyme that has two independent binding sites for a ligand, any inhibitor that binds solely to the free enzyme will give rise to positive co-operativity. 2. A model is considered for the allosteric control of enzymes by effectors in which their effects are mediated by ligand-induced perturbations of the ionization constants of a group or groups involved in the binding of substrate to the active site. 3. The model described offers a plausible explanation for the observation that the sigmoidal initial-rate curves reported for some regulatory enzymes are not expressed at all pH values where the enzyme is catalytically active.     

An inhibitor of host sigma-stimulated core enzyme activity that purifies with DNA-dependent RNA polymerase of E. coli following T4 phage infection

The content of the sigma subunit (as detected by gel electrophoresis) and activity with T4 DNA were examined with RNA polymerase fractions from both normal and T4 phage-infected $\text{E. coli}$. Sigma-containing fractions and core enzymes were obtained by phosphocellulose column chromatography. The sigma-containing fraction of the enzyme from infected cells, although somewhat stimulatory to both core enzymes alone, inhibits the normal sigma-stimulated activity of the core enzyme from infected cells at both low and high KCl concentration. Normal core enzyme activity is inhibited only at high KCl concentration.