Affinity labeling of adenylate kinase with adenosine diphosphopyridoxal. Presence of Lys21 in the ATP-binding site

Adenosine diphosphopyridoxal, the affinity labeling reagent specific for a lysyl residue in the nucleotide-binding site of several enzymes (Tagaya, M., and Fukui, T. (1986) Biochemistry 25, 2958-2964; Tamura, J. K., Rakov, R. D., and Cross R. L. (1986) J. Biol. Chem. 261, 4126-4133) was applied to adenylate kinase from rabbit muscle. Incubation of the enzyme with a low concentration of the reagent at 25 degrees C for 20 min followed by reduction by sodium borohydride resulted in rapid inactivation of the enzyme. Extrapolation to 100% loss of enzyme activity gave a value of 1.0 mol of the reagent per mol of enzyme. ADP, ATP, and MgATP almost completely protected the enzyme from inactivation, whereas AMP offered little retardation of the inactivation. Dilution of the inactivated enzyme which had not been treated with the reducing reagent led to restoration of enzyme activity. This reactivation was accelerated by ATP but not by AMP. Structural study of the labeled peptide showed that Lys21 is exclusively labeled by adenosine diphosphopyridoxal. These results suggest that the epsilon-amino group of Lys21 is located in the ATP-binding site of the enzyme, more specifically at or close to the subsite for the gamma-phosphate of the nucleotide.     

Why is one Bacillus alpha-amylase more resistant against irreversible thermoinactivation than another?

Half-lives of Bacillus alpha-amylases at 90 degrees C and pH 6.5 greatly increase in the series from Bacillus amyloliquefaciens to Bacillus stearothermophilus to Bacillus licheniformis, e.g. the difference in thermostability between the first and the third enzymes exceeds 2 orders of magnitude. This stabilization is achieved by lowering the rate constant of monomolecular conformational scrambling, which is the cause of irreversible thermoinactivation of B. amyloliquefaciens and B. stearothermophilus alpha-amylases, so that for B. licheniformis alpha-amylase, another process, deamidation of Asn/Gln residues, emerges as the cause of inactivation. The extra thermostability of the thermophilic enzyme was found to be mainly due to additional salt bridges involving a few specific lysine residues (Lys-385 and Lys-88 and/or Lys-253). These stabilizing electrostatic interactions reduce the extent of unfolding of the enzyme molecule at high temperatures, consequently making it less prone to forming incorrect (scrambled) structures and thus decreasing the overall rate of irreversible thermoinactivation. The implications of these findings for protein engineering are discussed.     

Thermodynamic,kinetic, and structural basis for recognition and repair of 8-oxoguanine in DNA by Fpg protein from Escherichia coli

X-ray analysis does not provide quantitative estimates of the relative importance of the molecular contacts it reveals or of the relative contributions of specific and nonspecific interactions to the total affinity of specific DNA to enzymes. Stepwise increase of DNA ligand complexity has been used to estimate the relative contributions of virtually every nucleotide unit of 8-oxoguanine-containing DNA to its total affinity for Escherichia coli 8-oxoguanine DNA glycosylase (Fpg protein). Fpg protein can interact with up to 13 nucleotide units or base pairs of single- and double-stranded ribo- and deoxyribo-oligonucleotides of different lengths and sequences through weak additive contacts with their internucleotide phosphate groups. Bindings of both single-stranded and double-stranded oligonucleotides follow similar algorithms, with additive contributions to the free energy of binding of the structural components (phosphate, sugar, and base). Thermodynamic models are provided for both specific and nonspecific DNA sequences with Fpg protein. Fpg protein interacts nonspecifically with virtually all of the base-pair units within its DNA-binding cleft: this provides approximately 7 orders of magnitude of affinity (Delta G degrees approximately equal to -9.8 kcal/mol) for DNA. In contrast, the relative contribution of the 8-oxoguanine unit of the substrate (Delta G degrees approximately equal to -0.90 kcal/mol) together with other specific interactions is <2 orders of magnitude (Delta G degrees approximately equal to -2.8 kcal/mol). Michaelis complex formation of Fpg protein with DNA containing 8-oxoguanine cannot of itself provide the major part of the enzyme specificity, which lies in the k(cat) term; the rate is increased by 6-8 orders of magnitude on going from nonspecific to specific oligodeoxynucleotides.     

Substrate channeling of oxalacetate in solid-state complexes of malate dehydrogenase and citrate synthase

Current evidence suggests that mitochondrial matrix enzymes exist in solid-state, multienzyme complexes in vivo. Addition of polyethylene glycol to a solution containing malate dehydrogenase and citrate synthase generates such a solid-state, enzyme complex in vitro at enzyme concentrations permitting kinetic measurements. Suspensions of the isolated, solid-state, hetero-complex of these enzymes were used to study the coupled reactions of citrate synthesis from malate, NAD, and CoASAc. The particles appear to be about 1 microgram in diameter. Considering the ratio of enzyme to oxalacetate molecules in or at the surface of the solid-state particles, one would expect oxalacetate to be converted to citrate within a few molecular distances of the site of oxalacetate generation. This model of "substrate channeling" (or alternatively a direct transfer of oxalacetate between enzymes) is supported by experiments with excess aspartate aminotransferase and glutamate added to the solution phase to give a reaction competing with the synthase for bulk phase oxalacetate. Quantities of aminotransferase that reduce the citrate reaction rate with soluble dehydrogenase and synthase by 90% do not significantly affect rates with comparable amounts of the dehydrogenase-synthase complex. We suggest that similar substrate channeling can occur in vivo and discuss the possible advantages provided thereby.     

Ornithine decarboxylase from Neurospora crassa. Purification, characterization, and regulation by inactivation

Ornithine decarboxylase, a highly regulated enzyme of the polyamine pathway, was purified 670-fold from mycelia of Neurospora crassa that were highly augmented for enzyme activity. The enzyme is significantly different from those reported from three other lower eucaryotic organisms: Saccharomyces cerevisiae, Physarum polycephalum, and Tetrahymena pyriformis. Instead, the enzyme closely resembles the enzymes from mammals. The Mr = 110,000 enzyme is a dimer of 53,000 Da subunits, with a specific activity of 2,610 mumol per h per mg of protein. Antisera were raised to the purified enzyme and were rendered highly specific by cross-absorption with extracts of a mutant strain lacking ornithine decarboxylase protein. With the antisera, we show that the inactivation of the enzyme in response to polyamines is proportional to the loss of ornithine decarboxylase protein over almost 2 orders of magnitude. This is similar to the inactivation process in certain mammalian tissues, and different from the process in S. cerevisiae and P. polycephalum, in which enzyme modification, without proportional loss of antigen, accompanies enzyme inactivation. The N. crassa enzyme is therefore suitable as a microbial model for studies of the molecular regulation of the mammalian enzyme.     

Dissecting a charged network at the active site of orotidine-5'-phosphate decarboxylase

The crystal structure of yeast orotidine-5'-phosphate decarboxylase in complex with the postulated transition state analog, 6-hydroxyuridine-5'-phosphate, reveals contacts between this inhibitor and a novel quartet of charged residues (Lys-59, Asp-91, Lys-93, and Asp-96) within the active site. The structure also suggests a possible interaction between O2 of the 6-hydroxyuridine-5'-phosphate pyrimidine ring and Gln-215. Here we report the results of mutagenesis of each of the charged active site residues and Gln-215. The activities of the Q215A and wild-type enzymes were equal indicating that any interactions between this residue and the pyrimidine ring are dispensable for efficient decarboxylation. For the D91A and K93A mutant enzymes, activity was reduced by more than 5 orders of magnitude and substrate binding could not be detected by isothermal calorimetry. For the D96A mutant enzyme, k(cat) was reduced by more than 5 orders of magnitude, and isothermal calorimetry indicated an 11-fold decrease in the affinity of this enzyme for the substrate in the ground state. For the K59A enzyme, k(cat) was reduced by a factor of 130, and K(m) had increased by a factor of 900. These results indicate that the integrity of the network of charged residues is essential for transition state stabilization.     

Possibilities of the method of step-by-step complication of ligand structure in studies of protein--nucleic acid interactions: mechanisms of functioning of some replication, repair, topoisomerization, and restriction enzymes

X-Ray structure analysis is one of the most informative methods for investigation of enzymes. However, it does not provide quantitative estimation of the relative efficiency of formation of contacts revealed by this method, and when interpreting the data this does not allow taking into account the relative contribution of some specific and nonspecific interactions to the total affinity of nucleic acids (NA) to enzymes. This often results in unjustified overestimation of the role of specific enzyme--NA contacts in affinity and specificity of enzyme action. In recent years we have developed new approaches to analysis of the mechanisms of protein--nucleic acid interactions allowing quantitative estimation of the relative contribution of virtually every nucleotide unit (including individual structural elements) to the total affinity of enzymes to long DNA and RNA molecules. It is shown that the interaction between enzymes and NA on the molecular level can be successfully analyzed by the methods of synthesis and analysis, that is, step-by-step simplification or complication of the structure of a long NA-ligand. This approach allows the demonstration that complex formation including formation of contacts between enzymes and specific NA units can provide neither high affinity of the enzymes to NA nor the specificity of their action. Using a number of sequence-independent replication and repair enzymes specifically recognizing a modified unit in DNA and also some sequence-dependent topoisomerization and restriction enzymes as examples, it was shown that virtually all nucleotide units within the DNA binding cleft interact with the enzyme, and high affinity mainly (up to 5-7 of 7-10 orders of magnitude) is provided by many weak additive interactions between these enzymes and various structural elements of the individual NA nucleotide units. At the same time, the relative contribution of specific interactions to the total affinity of NA is rather small and does not exceed 1-2 orders of magnitude. Specificity of enzyme action is provided by the stages of the enzyme-dependent NA adaptation to the optimal conformation and directly of catalysis: kcat increases by 3-7 orders of magnitude when changing from nonspecific to specific NA. In the present work we summarized our experience in studies of enzymes by the method of step-by-step complication of the ligand structure and performed a detailed analysis of the features of this approach and its possibilities for the study of protein--nucleic acid interactions on the molecular level.     

The mechanism of inactivation of lipoxygenases by acetylenic fatty acids

The inactivation of soybean lipoxygenase by 5,8,11,14-eicosatetraynoic acid was studied in detail. The inactivation was found to be time-dependent and irreversible. A kinetic scheme, based on the assumption of a rapid inactivation of the enzyme-product complex, yielded a Km value for 5,8,11,14-eicosatetraynoic acid of 1.3 microM, which is about a tenth of that described for arachidonic acid, and a reaction constant k+2 of 0.006s-1, which is four orders of magnitude lower. The reasons for these differences are discussed. Several types of experimental evidence indicate that the first step of the enzyme inactivation is the conversion of 5,8,11,14-eicosatetraynoic acid via a lipoxygenase reaction: (a) the conversion of radioactively labelled methyl ester of 5,8,11,14-eicosatetraynoic acid to other products; (b) the oxygen requirement of the inactivation; (c) the competitive protective effect of linoleic acid; (d) the similarity of the activation energy for both the dioxygenation of linoleic acid and the enzyme inactivation by 5,8,11,14-eicosatetraynoic acid; (e) the formation of one mole methionine sulfoxide/mole enzyme during the reaction with 5,8,11,14-eicosatetraynoic acid, similar to the suicidal reaction of reticulocyte lipoxygenase with 13LS-hydroperoxy-linoleic acid. These results, as well as the lack of covalent binding of 14C-labelled 5,8,11,14-eicosatetraynoic acid methyl ester, contradict the allene mechanism postulated by others [D.T. Downing, D.G. Ahern, and M. Bachta (1970) Biochem. Biophys. Res. Commun. 40, 218-223; K.H. Gibson (1977) Chem. Soc. Rev. 6, 489-510]. It is assumed that the susceptible methionine is located at the active centre of the enzyme.     

Inactivation of phospholipase A2 and metalloproteinase from Crotalus atrox venom by direct current

To achieve our aim of understanding the interactions between direct current and enzymes in solution, we exposed reconstituted Crotalus atrox venom to direct electric current by immersing two platinum thread electrodes connected to a voltage generator (between 0 and 8 V) into a reaction mixture for a few seconds. Then, we assayed the residual activity of phospholipases A(2) (PLA(2)),metalloproteinases, and phosphodiesterases, abundant in crotaline snake venoms and relevant in the pathophysiology of envenomation, characterized by hemorrhage, pain, and tissue damage. C. atrox venom phospholipase A(2) and metalloproteinases were consistently and irreversibly inactivated by direct current (between 0 and 0.7 mA) exposure. In contrast, C. atrox venom phosphodiesterases were not affected. Total protein content and temperature of the sample remained the same. Secretory pancreatic phospholipase A(2), homologue to snake venom phospholipases A(2), was also inactivated by direct current treatment. In order to understand the structural reasoning behind PLA(2) inactivation, circular dichroism measurements were conducted on homogeneous commercial pancreatic phospholipase A(2), and it was found that the enzyme undergoes structural alterations upon direct current exposure.     

Kinetic modeling of cellulose hydrolysis with first order inactivation of adsorbed cellulase

Enzymatic hydrolysis involves complex interaction between enzyme, substrate, and the reaction environment, and the complete mechanism is still unknown. Further, glucose release slows significantly as the reaction proceeds. A model based on Langmuir binding kinetics that incorporates inactivation of adsorbed cellulase was developed that predicts product formation within 10% of experimental results for two substrates. A key premise of the model, with experimental validation, suggests that V(max) decreases as a function of time due to loss of total available enzyme as adsorbed cellulases become inactivated. Rate constants for product formation and enzyme inactivation were comparable to values reported elsewhere. A value of k(2)/K(m) that is several orders of magnitude lower than the rate constant for the diffusion-controlled encounter of enzyme and substrate, along with similar parameter values between substrates, implies a common but undefined rate-limiting step associated with loss of enzyme activity likely exists in the pathway of cellulose hydrolysis.     

Comparison of fungal glucose oxidases. Chemical, physicochemical and immunological studies.

Glucose oxidases (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) from two fungal genera (Aspergillus and Penicillium) were studied chemically, physicochemically and immunologically to elucidate the similarities and dissimilarities between these enzymes. Investigation of circular dichroism spectra revealed that these enzymes proteins possess essentially identical conformations. However, differences found in thermal inactivation parameters, catalytic parameters and quantitative immunological reactivities indicate that these enzymes must have some minor but distinct variations in their structures. Interestingly, it was observed that the Penicillium enzyme cross-reacted with the antiserum against the Aspergillus enzyme with an association constant of two orders of magnitude lower than that of the Aspergillus enzyme, and that the precipitin one of the Penicillium enzyme fused together with that of the Aspergillus enzyme in the immunodouble diffusion test. These results lead to the conclusion that these enzymes are closely related but not completely identical, and suggest that they might have evolved from a common ancestral precursor.     

Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp-369-->Asn): a mechanism involving one zinc per active site.

Using site-directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km). The kcat for the D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme. The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p-nitrophenol phosphate was 2 orders of magnitude lower than for the wild-type enzyme. Thus the kcat/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp-369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group. The crystal structure of D369N, solved to 2.5 A resolution with an R-factor of 0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of the kinetic results and the refined X-ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.     

Particulate carbonic anhydrase in homogenates of human kidney.

About 2% of human kidney carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been found in particulate fractions. Its distribution in the particulate fractions obtained by differential centrifugation suggests that it may be concentrated in the brush border. The particulate enzyme is like red cell carbonic anhydrace C in its susceptibility to inhibition by anions. Particulate carbonic anhydrase is firmly bound to the membrane and is not released by incubation at pH 10.6 and 37 degrees C or by addition of Triton X-100 or deoxycholate. In 10% Triton X-100 at pH 11.3 and 37 degrees C, the particulate enzyme is inactivated with a half time of about 20 min, and this is at least an order of magnitude slower than the inactivation of soluble enzymes in the presence or absence of membranes. The soluble enzymes are inactivated within a few minutes at 25 degrees C in 3-4% sodium dodecyl sulfate, but the particulate enzyme is relatively stable under those conditions, and its half-time of inactivation at 14 degrees C with a detergent-protein ratio of 25 was about 24 h. Gel filtration with Ultragel AcA-44 in sodium dodecyl sulfate indicates that the membrane carbonic anhydrase has a molecular weight of less than 66 000, so its stability is not due to association with large membrane fragments or vesicles. These results suggest that the membrane enzyme may be a different isozyme than the soluble carbonic anhydrases. Although present in relatively small amounts, its localization on the membrane could give it functional significance.     

The role of weak specific and nonspecific interactions in recognition and conversation by enzymes of long DNA

According to a currently accepted model, enzymes engage in high-rate sliding along DNA when searching for specific recognition sequences or structural elements (modified nucleotides, breaks, single-stranded DNA fragments, etc.). Such sliding requires these enzymes to possess sufficiently high affinity for DNA of any sequence. Thus, significant differences in the enzymes' affinity for specific and nonspecific DNA sequences cannot be expected, and formation of a complex between an enzyme and its target DNA unlikely contributes significantly in the enzyme specificity. To elucidate the factors providing the specificity we have analyzed many DNA replication, DNA repair, topoisomerization, integration, and recombination enzymes using a number of physicochemical methods, including a method of stepwise increase in ligand complexity developed in our laboratory. It was shown that high affinity of all studied enzymes for long DNA is provided by formation of many weak contacts of the enzymes with all nucleotide units covered by protein globules. Contacts of positively charged amino acid residues with internucleotide phosphate groups contribute most to such interactions; the contribution of each contact is very small and the full contact interface usually resembles interactions between oppositely charged biopolymer surfaces. In some cases significant contribution to the affinity is made through hydrophobic and/or van der Waals interactions of the enzymes with nucleobases. Overall, depending on the enzyme, such nonspecific interactions provide 5-8 orders of the enzyme affinity for DNA. Specific interactions of enzymes with long DNA, in contrast to contacts of enzymes with small ligands, are usually weak and comparable in efficiency with weak nonspecific contacts. The sum of specific interactions most often provides approximately one and rarely two orders of the affinity. According to structural data, DNA binding to any of the investigated enzymes is followed by a stage of DNA conformation adjustment including partial or complete DNA melting, deformation of its backbone, stretching, compression, bending or kinking, eversion of nucleotides from the DNA helix, etc. The full set of such changes is characteristic for each individual enzyme. The fact that all enzyme-dependent changes in DNA are effected through weak specific rather than strong interactions is very important. Enzyme-specific changes in DNA conformation are required for effective adjustment of reacting orbitals with accuracy about 10-15 degrees, which is possible only for specific DNA. A transition from nonspecific to specific DNA leads to an increase in the reaction rate (kcat) by 4-8 orders of magnitude. Thus, the stages of DNA conformation adjustment and catalysis proper provide the high specificity of enzyme action.     

Thermodynamic, kinetic and structural basis for recognition and repair of abasic sites in DNA by apurinic/apyrimidinic endonuclease from human placenta

X-ray analysis of enzyme–DNA interactions is very informative in revealing molecular contacts, but provides neither quantitative estimates of the relative importance of these contacts nor information on the relative contributions of specific and nonspecific interactions to the total affinity of enzymes for specific DNA. A stepwise increase in the ligand complexity approach is used to estimate the relative contributions of virtually every nucleotide unit of synthetic DNA containing abasic sites to its affinity for apurinic/apyrimidinic endonuclease (APE1) from human placenta. It was found that APE1 interacts with 9–10 nt units or base pairs of single-stranded and double-stranded ribooligonucleotides and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleotide phosphate groups. Such nonspecific interactions of APE1 with nearly every nucleotide within its DNA-binding cleft provides up to seven orders of magnitude (ΔG° ~ −8.7 to −9.0 kcal/mol) of the enzyme affinity for any DNA substrate. In contrast, interactions with the abasic site together with other specific APE1–DNA interactions provide only one order of magnitude (ΔG° ~ −1.1 to −1.5 kcal/mol) of the total affinity of APE1 for specific DNA. We conclude that the enzyme's specificity for abasic sites in DNA is mostly due to a great increase (six to seven orders of magnitude) in the reaction rate with specific DNA, with formation of the Michaelis complex contributing to the substrate preference only marginally.     

Mobility of enzymes on insoluble substrates: the beta-amylase-starch gel system

The movement of enzymes along the surfaces of biopolymers containing enzyme-susceptible sites can be described as a lateral diffusion process characterized by an apparent diffusion coefficient [E. Katchalski-Katzir, J. Rishpon, E. Sahar, R. Lamed, and Y. I. Henis (1985) Biopolymers 24 , 257–277]. Studies on the diffusion of enzymes on biopolymer substrates can therefore provide important information on the mechanism of enzyme–biopolymer interaction. For this reason, the motion of fluorescently labeled β-amylase [α-D -(1 → 4)glucan maltohydrolase; E.C. 3.2.1.2] on the surface of starch gels was studied by fluorescence photobleaching recovery. The results indicate that the motion of β-amylase on the surface of the gel substrate occurs by both lateral diffusion along the surface (over micron distances) and exchange between bound and free enzyme molecules in the solution covering the gel, and that the two processes occur concomitantly and in a random manner. Surface diffusion also appears an important process with respect to the action of the enzyme on the substrate sites, since this component of the motion disappears upon inactivation of the enzyme, leaving only exchange to contribute to the measured motion.     

An affinity label for the regulatory dithiol of ribulose-5-phosphate kinase from maize (Zea mays).

Ribulose-5-phosphate kinase from maize (Zea mays) can exist in either a reduced, active form or an oxidized, inactive form. Reduced ribulose-5-phosphate kinase is rapidly and irreversibly inactivated by the dichlorotriazine dye Reactive Red 1 (Procion Red MX-2B), but the irreversible inactivation of the oxidized form of ribulose-5-phosphate kinase occurs at only 0.05% of this rate. The rate of inactivation of the reduced enzyme by Reactive Red 1 (apparent bimolecular rate constant 10(4)M-1 X s-1 at pH 7.4 and 25 degrees C) is several orders of magnitude greater than previous estimates of the rates of dye-mediated inactivation of other enzymes. The dye-dependent inactivation of the reduced enzyme is inhibited by Hg2+ or p-mercuribenzoate (thiol reagents that reversibly inhibit ribulose-5-phosphate kinase activity), or by ATP and ADP, the nucleotide substrates of the enzyme. Hydrolysed Reactive Red 1, which does not inactivate the enzyme, is a reversible inhibitor of ribulose-5-phosphate kinase. This inhibition is competitive with respect to ATP (Ki approximately 0.5 mM). The dye appears to act as an affinity label for the ATP/ADP-binding site by preferentially arylating a thiol residue generated during the reductive activation of the enzyme that is achieved by dithiothreitol or thioredoxin in vitro or during illumination of leaves.     

Post-irradiation inactivation,protection, and repair of the sulfhydryl enzyme malate synthase

Malate synthase from baker's yeast, a trimeric sulfhydryl enzyme with one essential sulfhydryl group per subunit, was inactivated by 2 kGy X-irradiation in air-saturated aqueous solution (enzyme concentration: 0.5 mg/ml). The radiation induced changes of enzymic activity were registered at about 0, 30, 60 h after irradiation. To elucidate the role of OH., O-.2, and H2O2 in the X-ray inactivation of the enzyme, experiments were performed in the absence or presence of different concentrations of specific additives (formate, superoxide dismutase, catalase). These additives were added to malate synthase solutions before or after X-irradiation. Moreover, repairs of inactivated malate synthase were initiated at about 0 or 30 h after irradiation by means of the sulfhydryl agent dithiothreitol. Experiments yielded the following results: Irradiation of malate synthase in the absence of additives inactivated the enzyme immediately to a residual activity Ar = 3% (corresponding to a D37 = 0.6 kGy), and led to further slow inactivation in the post-irradiation phase. Repairs, initiated at different times after irradiation, restored enzymic activity considerably. The repair initiated at t = 0 led to Ar = 21%; repairs started later on resulted in somewhat lower activities. The decay of repairability, however, was found to progress more slowly than post-irradiation inactivation itself. After completion of repair the activities of repaired samples did not decrease significantly. The presence of specific additives during irradiation caused significant protective effects against primary inactivation. The protection by formate was very pronounced (e.g., Ar = 72% and D37 = 6 kGy for 100 mM formate). The presence of catalytic amounts of superoxide dismutase and/or catalase exhibited only minor effects, depending on the presence and concentration of formate. Both the presence of specific additives during irradiation and the addition of additives after irradiation may alter the post-irradiation inactivation. Catalase turned out to be the most potent inhibitor of post-irradiation inactivation; superoxide dismutase showed an ambivalent behaviour, it accelerated or impeded post-irradiation inactivation; formate, when added after irradiation, exhibited a moderate protective effect. The presence of specific additives, added before and/or after irradiation, influenced the repair behaviour to some extent. The highest activity achieved by repair amounted to about 90% of the activity of the corresponding unirradiated sample. The percentual gain of activity was found to be the greater the lower the residual activity of the enzyme was before initiation of repair.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel non-heme iron-containing dioxygenase. Chloridazon-catechol dioxygenase from Phenylobacterium immobilis DSM 1986

Previously we purified an enzyme from Phenylobacterium immobilis DSM 1986, which cleaves the catechol derivative of the herbicide Chloridazon [5-amino-4-chloro-2-phenyl-3 (2H)-pyridazinone] in the meta position. The enzyme, which could be crystallized, proved in Ouchterlony double-diffusion tests to consist of a single protein species. No cross-reaction was observed with other meta-cleaving enzymes. Its light absorption spectrum showed a maximum at 279 nm (epsilon = 310 mM -1 cm -1), shoulders at 289 nm and 275 nm and a very weak band at around 430 nm (epsilon = 1.14 mM -1 cm -1). The amino acid analysis showed a slight excess of acidic amino acids, in agreement with the pl of 4.5. Surprisingly the enzyme per se is completely inactive, although it contains one non-dialysable iron atom per submit. It has to be activated by preincubation with ferrous ions or ascorbate. The enzyme activated this way is autoxidizable and returns to its non-activated state in the presence of oxygen. During the reaction with the substrate, this inactivation seems to be enhanced about 100 times. Since this kind of activation and inactivation is not observed in other meta-cleaving enzymes, this enzyme seems to represent a new type of a non-heme iron dioxygenase. We tentatively propose the name Chloridazon-catechol dioxygenase for this enzyme.     

Inhibition of venom phospholipases A2 by manoalide and manoalogue. Stoichiometry of incorporation

We have previously described the irreversible inhibition of cobra venom phospholipase A2 (PLA2) by the marine natural product manoalide (MLD) (Lombardo, D., and Dennis, E. A. (1985) J. Biol. Chem. 260, 7234-7240) and by its synthetic analog, manoalogue (MLG) (Reynolds L. J., Morgan, B. P., Hite, G. A., Mihelich, E. D., and Dennis, E. A. (1988) J. Am. Chem. Soc. 110, 5172-5177). We have now made a direct comparison of the action of these two inhibitors on PLA2 from cobra, bee, and rattlesnake venoms and have found that MLG behaves kinetically similarly to MLD in all cases with only minor differences. The time courses of inactivation differ significantly between the three enzymes, however, with the inactivation of bee and rattlesnake PLAs2, occurring much faster than does the inactivation of the cobra venom enzyme. The enzymes also differ in their sensitivity to the presence of Ca2+ during the inactivation. Of the three enzymes, the most Ca(2+)-sensitive is the rattlesnake enzyme, which shows a much faster rate of inactivation in the presence of Ca2+ than in the presence of EGTA. However, the same rate of inactivation was also observed when the inhibitor Ba2+ was substituted for Ca2+, indicating that catalytic activity is not required for inactivation of the enzyme. To probe the mechanism of inactivation and to determine the stoichiometry of incorporation, we have synthesized 3H-labeled MLG and have found that inactivation of cobra PLA2 is accompanied by an incorporation of 3.8 mol of [3H]MLG/mol of enzyme. The same amount of 3H incorporation was observed when p-bromophenacyl bromide-inactivated PLA2 was incubated with [3H]MLG, again indicating that catalytic activity is not required for the reaction of PLA2 with MLG. All together, these results suggest that MLD and MLG are not suicide inhibitors of PLA2. A portion of the incorporated radioactivity was acid-labile, and dialysis of the radiolabeled PLA2 under acidic conditions resulted in a loss of about one-third of the enzyme-associated radioactivity, leaving 2.4 mol of [3H]MLG/mol of PLA2. In previous studies, amino acid analysis, which also included acid treatment, indicated that MLG-modified cobra phospholipase A2 contained 2.8 mol of Lys less than the native enzyme. Thus, 1 mol of [3H]MLG is incorporated per mol of Lys lost. The implications of this 1:1 stoichiometry of MLG to Lys on the mechanism of reaction of these inhibitors is discussed.