Purification, characterization, and kinetic mechanism of S-adenosyl-L-methionine:macrocin O-methyltransferase from Streptomyces fradiae

S-Adenosyl-L-methionine:macrocin O-methyltransferase catalyzes conversion of macrocin to tylosin, the terminal and main rate-limiting step of tylosin biosynthesis in Streptomyces fradiae. The O-methyltransferase was stabilized in vitro and purified to electrophoretic homogeneity. The purified enzyme had a molecular weight of 65,000 and consisted of two identical subunits of 32,000 with an isoelectric point of 4.5. The enzyme required Mg2+, Mn2+, or Co2+ for maximal activity and was catalytically optimal at pH 7.5-8.0 and 31 degrees C. The O-methyltransferase catalyzed the conversion of macrocin to tylosin at a stoichiometric ratio of 1:1. The enzyme also mediated conversion of lactenocin----desmycosin. The corresponding Vmax/Km ratios for the two analogous conversions were similar, and both enzymic conversions were susceptible to extensive competitive and noncompetitive inhibitions by macrolide metabolites. Steady-state kinetic studies for initial velocity, substrate analogue, and product inhibitions have allowed formulation of Ordered Bi Bi as the reaction mechanism for macrocin O-methyltransferase.     

Purification, properties, and kinetics of D-ribulokinase from Aerobacter aerogenes.

The enzyme D-ribulokinase from Aerobacter aerogenes was purified to near homogeneity. The molecular weight, as determined by Sephacryl gel chromatography, is 116,000. The subunit molecular weight, determined by sodium dodecyl sulfate-gel electrophoresis, is 59,000, suggesting that D-ribulokinase is a dimer of identical subunits. Initial rate kinetic studies, involving substrate analogs and products, were carried out. These investigations support a kinetic mechanism of the Random Bi Bi type. Isotope partitioning, utilizing D-[3H]ribulose, indicates that the mechanism is steady state Random Bi Bi.     

The effect of bulk hydrogen ion concentration upon the apparent kinetic parameters of purified pig liver catechol O-methyltransferase

In order to investigate the pH dependence of catechol O-methyltransferase (S-adenosyl-L-methionine:catechol O-methyltransferase, EC 2.1.1.6), kinetic parameters have been determined for the highly purified enzyme from pig liver over the pH range 6.75-8.20 using the substrates S-adenosylmethionine (AdoMet) and 3,4-dihydroxyphenylacetic acid (DOPAC). The Km for AdoMet was found to be invariant with pH while the Km for DOPAC decreased sharply with increasing pH. The group responsible for the latter has a pK of approx. 7.1. The logarithmic (Dixon) plot of Km against pH for both substrates and that of Vmax/Km against pH for DOPAC mirror the kinetic behaviour revealed by linear plots. However, for other parameters, linear graphs indicate peaks too narrow to be explicable by a simple kinetic mechanism, whereas logarithmic plots of these parameters produce graphs apparently not reflecting this behaviour. We conclude that these results are not the products of random error or artefactual data analysis but are too complex to be explicable by a simple model of kinetic behaviour. Possible explanations (adherence of catechol O-methyltransferase to a higher-order mechanism or a dual mode of substrate binding) are advanced.     

Molecular characterization of the S-adenosyl-L-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase involved in isoquinoline alkaloid biosynthesis in Coptis japonica

S-adenosyl-L-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'-OMT) catalyzes the conversion of 3'-hydroxy-N-methylcoclaurine to reticuline, an important intermediate in synthesizing isoquinoline alkaloids. In an earlier step in the biosynthetic pathway to reticuline, another O-methyltransferase, S-adenosyl-L-methionine:norcoclaurine 6-O-methyltransferase (6-OMT), catalyzes methylation of the 6-hydroxyl group of norcoclaurine. We isolated two kinds of cDNA clones that correspond to the internal amino acid sequences of a 6-OMT/4'-OMT preparation from cultured Coptis japonica cells. Heterologously expressed proteins had 6-OMT or 4'-OMT activities, indicative that each cDNA encodes a different enzyme. 4'-OMT was purified using recombinant protein, and its enzymological properties were characterized. It had enzymological characteristics similar to those of 6-OMT; the active enzyme was the dimer of the subunit, no divalent cations were required for activity, and there was inhibition by Fe(2+), Cu(2+), Co(2+), Zn(2+), or Ni(2+), but none by the SH reagent. 4'-OMT clearly had different substrate specificity. It methylated (R,S)-6-O-methylnorlaudanosoline, as well as (R, S)-laudanosoline and (R,S)-norlaudanosoline. Laudanosoline, an N-methylated substrate, was a much better substrate for 4'-OMT than norlaudanosoline. 6-OMT methylated norlaudanosoline and laudanosoline equally. Further characterization of the substrate saturation and product inhibition kinetics indicated that 4'-OMT follows an ordered Bi Bi mechanism, whereas 6-OMT follows a Ping-Pong Bi Bi mechanism. The molecular evolution of these two related O-methyltransferases is discussed.     

Purification and kinetics of sheep kidney cortex glucose-6-phosphate dehydrogenase

Glucose-6-phosphate dehydrogenase (G-6-PD) is one of the important enzymes, which is responsible for the production of NADPH and ribose-5-phosphate. NADPH is used for the biosynthetic reactions and protection of the cells from free radicals. We have investigated some properties and kinetic mechanism of the sheep kidney cortex G-6-PD. This enzyme has been purified 1,384-fold with a yield of 16.96% and had a specific activity of 27.69 U/mg protein. The purification procedure consists of 2', 5'-ADP-Sepharose 4B affinity chromatography after ultracentrifugation. The sheep kidney cortex G-6-PD was found to operate according to a Ping Pong Bi Bi mechanism. The kinetic parameters from sheep K(m) values for G-6-P and NADP(+) and V(m) were determined to be 0.041+/-0.0043 mM, 0.0147+/-0.001 mM and 28.23+/-0.86 microMol min(-1) mg protein(-1), respectively. The pH optimum was 7.4 and the optimum temperature was 45 degrees C. In our previous study we have found that lamb kidney cortex G-6-PD enzyme obeys 'Ordered Bi Bi' mechanism. We suggest that kinetic mechanism altered due to the aging since sheep G-6-PD uses a 'ping pong' mechanism.     

Purification and characterization of the maize amyloplast stromal 112-kDa starch phosphorylase

A plastidic 112-kDa starch phosphorylase (SP) has been identified in the amyloplast stromal fraction of maize. This starch phosphorylase was purified 310-fold from maize endosperm and characterized with respect to its enzymological and kinetic properties. The purification procedure included ammonium sulfate fractionation, Sephacryl 300 HR chromatography, affinity starch adsorption, Q-Sepharose, and Mono Q chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and SDS-polyacrylamide gel electrophoresis. Anti-SP antibodies recognized the purified 112-kDa SP enzyme and N-terminal amino acid sequence analysis confirmed that the purified enzyme is the amyloplast stromal 112-kDa SP. Analysis of the purified enzyme by Superose 6 gel filtration chromatography indicated that the native enzyme consisted of two identical subunits. The pH optimum for the enzyme was 6.0 in the synthetic direction and 5.5 in the phosphorolytic direction. SP activity was inhibited by thioreactive agents, diethyl pyrocarbonate, phenylglyoxal, and ADP-glucose. The activation energies for the synthetic and phosphorolytic reactions were 11.1 and 16.9 kcal/mol, respectively, and the enzyme was thermally labile above 50 degrees C. Results of kinetic experiments indicated that the enzyme catalyzes its reaction via a sequential Bi Bi mechanism. The Km value for amylopectin was eight-fold lower than that of glycogen. A kinetic analysis indicated that the phosphorolytic reaction was favored over the synthetic reaction when malto-oligosaccharides (4 to 7 units) were used as substrates. The specificity constants (Vmax/Km) of the enzyme measured in either the synthetic or the phosphorolytic directions increased with increasing chain length.     

Substrate specificity and kinetic mechanism of mammalian G9a histone H3 methyltransferase

Lysine-specific murine histone H3 methyltransferase, G9a, was expressed and purified in a baculovirus expression system. The primary structure of the recombinant enzyme is identical to the native enzyme. Enzymatic activity was favorable at alkaline conditions (>pH 8) and low salt concentration and virtually unchanged between 25 and 42 degrees C. Purified G9a was used for substrate specificity and steady-state kinetic analysis with peptides representing un- or dimethylated lysine 9 histone H3 tails with native lysine 4 or with lysine 4 changed to alanine (K4AK9). In vitro methylation of the H3 tail peptide resulted in trimethylation of Lys-9 and the reaction is processive. The turnover number (k(cat)) for methylation was 88 and 32 h(-1) on the wild type and K4AK9 histone H3 tail, respectively. The Michaelis constants for wild type and K4AK9 ((K(m)(pep))) were 0.9 and 1.0 microM and for S-adenosyl-L-methionine (K(m)(AdoMet)) were 1.8 and 0.6 microM, respectively. Comparable kinetic constants were obtained for recombinant histone H3. The conversion of K4AK9 di- to trimethyl-lysine was 7-fold slower than methyl group addition to unmethylated peptide. Preincubation studies showed that G9a-AdoMet and G9a-peptide complexes are catalytically active. Initial velocity data with peptide and S-adenosyl-L-methionine (AdoMet) and product inhibition studies with S-adenosyl-L-homocysteine were performed to assess the kinetic mechanism of the reaction. Double reciprocal plots and preincubation studies revealed S-adenosyl-L-homocysteine as a competitive inhibitor to AdoMet and mixed inhibitor to peptide. Trimethylated peptides acted as a competitive inhibitor to substrate peptide and mixed inhibitor to AdoMet suggesting a random mechanism in a Bi Bi reaction for recombinant G9a where either substrate can bind first to the enzyme, and either product can release first.     

Pig liver phosphomevalonate kinase: kinetic mechanism

Phosphomevalonate kinase catalyzes the phosphorylation of phosphomevalonate to diphosphomevalonate by ATP, one of the initial steps in the biosynthesis of steroids and isoprenoids. In previous studies, the enzyme from pig liver was purified and characterized, and preliminary work showed that the enzyme follows hyperbolic kinetics and a sequential mechanism. The present work is a more detailed analysis of its kinetic mechanism, using initial velocity and isotope exchange at equilibrium measurements. The results are compatible with a Bi Bi sequential ordered mechanism with phosphomevalonate as the first substrate and ADP the last product. The Km values estimated are 43+/-7 microM for Mg-ATP and 12+/-3 microM for phosphomevalonate, with a Vmax of 51+/-2 micromol min-1 mg of protein-1.     

Continuous-culture studies on the regulation of tylosin biosynthesis

The metabolic regulation of tylosin synthesis by Streptomyces fradiae NRRL 2702 was studied in batch and chemostat cultures using a soluble synthetic medium. In batch culture a medium which diminished the trophophase-idiophase kinetic pattern was used to assess the activities of the enzymes involved in tylosin synthesis. The enzymes methylmalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1) and propionyl-coenzyme A carboxylase (EC 6.4.1.3) showed early enzymatic derepression, both enzymes reaching their highest specific activities after 72-96 fermentation. The activity of macrocin 3' -O-methyltransferase, the enzyme catalyzing the conversion of macrocin (tylosin C) to tylosin (tylosin A). also peaked at 72 h. The specific activities of the three enzymes showed close correlation with the q(tylosin) value. In chemostat cultures the activities of the enzymes and the intracellular level of the adenylate pool and energy charge were studied as a function of dilution rate. Under steady-state conditions, increases in the specific growth rate repressed the enzymes activities with a concomitant increase in the intracellular level of the adenylate pool, while the adenylate energy charge remained almost constant and in the range 0.5-0.52. The highest specific activities of the enzymes were observed when D = 0.008 h (-1). The specific rate of tylosin synthesis was inversely proportional to the specific growth rate and the intracellular level of adenylate pool. The pool of adenylate could be a nutritional parameter which had a considerable influence on the biosynthesis of tylosin.     

Isocitrate lyase from Phycomyces blakesleeanus. The role of Mg2+ ions, kinetics and evidence for two classes of modifiable thiol groups.

Isocitrate lyase was purified from Phycomyces blakesleeanus N.R.R.L. 1555(-). The native enzyme has an Mr of 240,000. The enzyme appeared to be a tetramer with apparently identical subunits of Mr 62,000. The enzyme requires Mg2+ for activity, and the data suggest that the Mg2(+)-isocitrate complex is the true substrate and that Mg2+ ions act as a non-essential activator. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated an ordered Uni Bi mechanism and the kinetic constants of the model were calculated. The spectrophotometric titration of thiol groups in Phycomyces isocitrate lyase with 5.5'-dithiobis-(2-nitrobenzoic acid) gave two free thiol groups per subunit of enzyme in the native state and three in the denatured state. The isocitrate lyase was completely inactivated by iodoacetate, with non-linear kinetics. The inactivation data suggest that the enzyme has two classes of modifiable thiol groups. The results are also in accord with the formation of a non-covalent enzyme-inhibitor complex before irreversible modification of the enzyme. Both the equilibrium constants for formation of the complex and the first-order rate constants for the irreversible modification step were determined. The partial protective effect of isocitrate and Mg2+ against iodoacetate inactivation was investigated in a preliminary form.     

Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae

The membrane-associated phospholipid biosynthetic enzyme CDP-diacylglycerol synthase (CTP:phosphatidate cytidylyltransferase, EC 2.7.7.41) was purified 2,300-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of mitochondrial membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and hydroxylapatite chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiation inactivation of mitochondrial associated and purified CDP-diacylglycerol synthase suggested that the molecular weight of the native enzyme was 114,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme preparation yielded two subunits with molecular weights of 56,000 and 54,000. Antibodies prepared against the purified enzyme immunoprecipitated CDP-diacylglycerol synthase activity and subunits. CDP-diacylglycerol synthase activity was dependent on magnesium ions and Triton X-100 at pH 6.5. Thio-reactive agents inhibited activity. The activation energy for the reaction was 9 kcal/mol, and the enzyme was thermally labile above 30 degrees C. The Km values for CTP and phosphatidate were 1 and 0.5 mM, respectively, and the Vmax was 4,700 nmol/min/mg. Results of kinetic and isotopic exchange reactions suggested that the enzyme catalyzes a sequential Bi Bi reaction mechanism.     

Cloning and characterization of a cDNA from Aspergillus parasiticus encoding an O-methyltransferase involved in aflatoxin biosynthesis.

Aflatoxins are polyketide-derived secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Among the catalytic steps in the aflatoxin biosynthetic pathway, the conversion of sterigmatocystin to O-methylsterigmatocystin and the conversion of dihydrosterigmatocystin to dihydro-O-methylsterigmatocystin are catalyzed by an S-adenosylmethionine-dependent O-methyltransferase. A cDNA library was constructed by using RNA isolated from a 24-h-old culture of wild-type A. parasiticus SRRC 143 and was screened by using polyclonal antiserum raised against a purified 40-kDa O-methyltransferase protein. A clone that harbored a full-length cDNA insert (1,460 bp) containing the 1,254-bp coding region of the gene omt-1 was identified by the antiserum and isolated. The complete cDNA sequence was determined, and the corresponding 418-amino-acid sequence of the native enzyme with a molecular weight of 46,000 was deduced. This 46-kDa native enzyme has a leader sequence of 41 amino acids, and the mature form of the enzyme apparently consists of 377 amino acids and has a molecular weight of 42,000. Direct sequencing of the purified mature enzyme from A. parasiticus SRRC 163 showed that 19 of 22 amino acid residues were identical to the amino acid residues in an internal region of the deduced amino acid sequence of the mature protein. The 1,460-bp omt-1 cDNA was cloned into an Escherichia coli expression system; a Western blot (immunoblot) analysis of crude extracts from this expression system revealed a 51-kDa fusion protein (fused with a 5-kDa beta-galactosidase N-terminal fragment).(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic mechanism and enantioselectivity of halohydrin dehalogenase from Agrobacterium radiobacter

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the reversible intramolecular nucleophilic displacement of a halogen by a hydroxyl group in vicinal haloalcohols, producing the corresponding epoxides. The enzyme displays high enantioselectivity toward some aromatic halohydrins. To understand the kinetic mechanism and enantioselectivity of the enzyme, steady-state and pre-steady-state kinetic analysis was performed with p-nitro-2-bromo-1-phenylethanol (PNSHH) as a model substrate. Steady-state kinetic analyses indicated that the k(cat) of the enzyme with the (R)-enantiomer (22 s(-1)) is 3-fold higher than with the (S)-enantiomer and that the K(m) for the (R)-enantiomer (0.009 mM) is about 45-fold lower than that for the (S)-enantiomer, resulting in a high enantiopreference for the (R)-enantiomer. Product inhibition studies revealed that HheC follows an ordered Uni Bi mechanism for both enantiomers, with halide as the first product to be released. To identify the rate-limiting step in the catalytic cycle, pre-steady-state experiments were performed using stopped-flow and rapid-quench methods. The results revealed the existence of a pre-steady-state burst phase during conversion of (R)-PNSHH, whereas no such burst was observed with the (S)-enantiomer. This indicates that a product release step is rate-limiting for the (R)-enantiomer but not for the (S)-enantiomer. This was further examined by doing single-turnover experiments, which revealed that during conversion of the (R)-enantiomer the rate of bromide release is 21 s(-1). Furthermore, multiple turnover analyses showed that the binding of (R)-PNSHH is a rapid equilibrium step and that the rate of formation of product ternary complex is 380 s(-1). Taken together, these findings enabled the formulation of an ordered Uni Bi kinetic mechanism for the conversion of (R)-PNSHH by HheC in which all of the rate constants are obtained. The high enantiopreference for the (R)-enantiomer can be explained by weak substrate binding of the (S)-enantiomer and a lower rate of reaction at the active site.     

Kinetic mechanism of glucose dehydrogenase from Halobacterium salinarum

The kinetic mechanism of glucose dehydrogenase (EC 1.1.1.47) from Halobacterium salinarum was studied by initial velocity and product inhibition methods. The results suggest that both, in the forward and reverse direction, the reaction mechanism is of Bi Bi sequential ordered type involving formation of ternary complexes. NADP+ adds first and NADPH formed dissociates from the enzyme last. For the reverse direction, NADPH adds first and NADP+ leaves last. Product inhibition experiments indicate that (a), the coenzymes compete for the same site and form of the enzyme and (b), ternary abortive complexes of enzyme-NADP(+)-glucono-delta-lactone and enzyme-NADPH-glucose are formed. All the other inhibitions are noncompetitive.     

Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids

Coenzyme A (CoA) transferase from Clostridium acetobutylicum ATCC 824 was purified 81-fold to homogeneity. This enzyme was stable in the presence of 0.5 M ammonium sulfate and 20% (vol/vol) glycerol, whereas activity was rapidly lost in the absence of these stabilizers. The kinetic binding mechanism was Ping Pong Bi Bi, and the Km values at pH 7.5 and 30 degrees C for acetate, propionate, and butyrate were, respectively, 1,200, 1,000, and 660 mM, while the Km value for acetoacetyl-CoA ranged from about 7 to 56 microM, depending on the acid substrate. The Km values for butyrate and acetate were high relative to the intracellular concentrations of these species; consequently, in vivo enzyme activity is expected to be sensitive to changes in those concentrations. In addition to the carboxylic acids listed above, this CoA transferase was able to convert valerate, isobutyrate, and crotonate; however, the conversion of formate, n-caproate, and isovalerate was not detected. The acetate and butyrate conversion reactions in vitro were inhibited by physiological levels of acetone and butanol, and this may be another factor in the in vivo regulation of enzyme activity. The optimum pH of acetate conversion was broad, with at least 80% of maximal activity from pH 5.9 to greater than 7.8. The purified enzyme was a heterotetramer with subunit molecular weights of about 23,000 and 25,000.     

Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids.

Coenzyme A (CoA) transferase from Clostridium acetobutylicum ATCC 824 was purified 81-fold to homogeneity. This enzyme was stable in the presence of 0.5 M ammonium sulfate and 20% (vol/vol) glycerol, whereas activity was rapidly lost in the absence of these stabilizers. The kinetic binding mechanism was Ping Pong Bi Bi, and the Km values at pH 7.5 and 30 degrees C for acetate, propionate, and butyrate were, respectively, 1,200, 1,000, and 660 mM, while the Km value for acetoacetyl-CoA ranged from about 7 to 56 microM, depending on the acid substrate. The Km values for butyrate and acetate were high relative to the intracellular concentrations of these species; consequently, in vivo enzyme activity is expected to be sensitive to changes in those concentrations. In addition to the carboxylic acids listed above, this CoA transferase was able to convert valerate, isobutyrate, and crotonate; however, the conversion of formate, n-caproate, and isovalerate was not detected. The acetate and butyrate conversion reactions in vitro were inhibited by physiological levels of acetone and butanol, and this may be another factor in the in vivo regulation of enzyme activity. The optimum pH of acetate conversion was broad, with at least 80% of maximal activity from pH 5.9 to greater than 7.8. The purified enzyme was a heterotetramer with subunit molecular weights of about 23,000 and 25,000.     

Characterization of the propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica: residue Lys592 is required for propionyl-AMP synthesis

The propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica catalyzes the first step of propionate catabolism, i.e., the activation of propionate to propionyl-CoA. The PrpE enzyme was purified, and its kinetic properties were determined. Evidence is presented that the conversion of propionate to propionyl-CoA proceeds via a propionyl-AMP intermediate. Kinetic experiments demonstrated that propionate was the preferred acyl substrate (kcat/Km = 1644 mM(-1) x s(-1)). Adenosine 5'-propyl phosphate was a potent inhibitor of the enzyme, and inhibition kinetics identified a Bi Uni Uni Bi Ping Pong mechanism for the reaction catalyzed by the PrpE enzyme. Site-directed mutagenesis was used to change the primary sequence of the wild-type protein at positions G245A, P247A, K248A, K248E, G249A, K592A, and K592E. Mutant PrpE proteins were purified, and the effects of the mutations on enzyme activity were investigated. Both PrpEK592 mutant proteins (K592A and K592E) failed to convert propionate to propionyl-CoA, and plasmids containing these alleles of prpE failed to restore growth on propionate of S. enterica carrying null prpE alleles on their chromosome. Both PrpEK592 mutant proteins converted propionyl-AMP to propionyl-CoA, suggesting residue K592 played no discernible role in thioester bond formation. To the best of our knowledge, these mutant proteins are the first acyl-CoA synthetases reported that are defective in adenylation activity.     

Properties of catechol O-methyltransferases from brain and liver of rat and human.

Kinetic and electrophoretic properties of catechol O-methyltransferases (EC 2.1.1.6) from brain and liver were studied. The enzyme of either rat or human tissues exhibited a single molecular form when subjected to electrophoresis at pH7.9. At pH9 a second, apparently oxidized, form was detected. Isoelectric-focusing experiments also indicated only one enzyme form, which was identical from extracts of brain and liver of each species (pI = 5.2 for rat, 5.5 for human). Similarities between brain and liver catechol O-methyltransferase of a given species were also demonstrated by kinetic parameters, meta/para ratios of products, and inhibitor potencies. Human catechol O-methyltransferase exhibited lower Km values than did the rat enzyme for S-adenosyl-L-methionine, dopamine and dihydroxybenzoic acid. Adrenochrome inhibited both rat and human enzyme. It was concluded (1) that only a single enzyme form could be demonstrated in the physiological pH region; (2) that catechol O-methyltransferase of brain could not be distinguished from the liver enzyme of the same species; and (3) that species differences exist between the enzymes of rat and human tissues.     

S-adenosylmethionine: protein-arginine methyltransferase. Purification and mechanism of the enzyme.

Protein methylase I (S-adenosylmethionine: protein-arginine methyltransferase, EC 2.1.1.23) has been purified from calf brain approximately 120-fold with a 14% yield. The final preparation is completely free of any other protein-specific methyltransferases and endogenous substrate protein. The enzyme has an optimum pH of 7.2 and pI value of 5.1. The Km values for S-adenosyl-L-methionine, histone H4, and an ancephalitogenic basic protein are 7.6 X 10(-6), 2.5 X 10(-5), and 7.1 X 10(-5) M, respectively, and the Ki value for S-adenosyl-L-homocysteine is 2.62 X 10(-6) M. The enzyme is highly specific for the arginine residues of protein, and the end products after hydrolysis of the methylated protein are NG,NG-di(asymmetric), NG,N'G-di(symmetric), and NG-monomethylarginine. The ratio of [14C]methyl incorporation into these derivatives by enzyme preparation at varying stages of purification remains unchanged at 40:5:55, strongly indicating that a single enzyme is involved in the synthesis of the three arginine derivatives. The kinetic mechanism of the protein methylase I reaction was studied with the purified enzyme. Initial velocity patterns converging at a point on the extended axis of abscissas were obtained with either histone H4 or S-adenosyl-L-methionine as the varied substrate. Product inhibition by S-adenosyl-L-homocysteine with S-adenosyl-L-methionine as the varied substrate was competitive regardless of whether or not the enzyme was saturated with histone H4. On the other hand, when histone H4 is the variable substrate, noncompetitive inhibition was obtained with S-adenosyl-L-homocysteine under conditions where the enzyme is not saturated with the other substrate, S-adenosyl-L-methionine. These results suggest that the mechanism of the protein methylase I reaction is a Sequential Ordered Bi Bi mechanism with S-adenosyl-L-methionine as the first substrate, histone H4 as the second substrate, methylated histone H4 as the first product, and S-adenosyl-L-homocysteine as the second product released.     

7,8-Diaminoperlargonic acid aminotransferase from Mycobacterium tuberculosis, a potential therapeutic target. Characterization and inhibition studies

Diaminopelargonic acid aminotransferase (DAPA AT), which is involved in biotin biosynthesis, catalyzes the transamination of 8-amino-7-oxononanoic acid (KAPA) using S-adenosyl-l-methionine (AdoMet) as amino donor. Mycobacterium tuberculosis DAPA AT, a potential therapeutic target, has been overproduced in Escherichia coli and purified to homogeneity using a single efficient step on a nickel-affinity column. The enzyme shows an electronic absorption spectrum typical of pyridoxal 5'-phosphate-dependent enzymes and behaves as a homotetramer in solution. The pH profile of the activity at saturation shows a single ionization group with a pK(a) of 8.0, which was attributed to the active-site lysine residue. The enzyme shows a Ping Pong Bi Bi kinetic mechanism with strong substrate inhibition with the following parameters: K(mAdoMet) = 0.78 +/- 0.20 mm, K(mKAPA) = 3.8 +/- 1.0 microm, k(cat) = 1.0 +/- 0.2 min(-1), K(iKAPA) = 14 +/- 2 microm. Amiclenomycin and a new analogue, 4-(4c-aminocyclohexa-2,5-dien-1r-yl)propanol (referred to as compound 1), were shown to be suicide substrates of this enzyme, with the following inactivation parameters: K(i) = 12 +/- 2 microm, k(inact) = 0.35 +/- 0.05 min(-1), and K(i) = 20 +/- 2 microm, k(inact) = 0.56 +/- 0.05 min(-1), for amiclenomycin and compound 1, respectively. The inactivation was irreversible, and the partition ratios were 1.0 and 1.1 for amiclenomycin and compound 1, respectively, which make these inactivators particularly efficient. compound 1 (100 microg.mL(-1)) completely inhibited the growth of an E. coli C268bioA mutant strain transformed with a plasmid expressing the M. tuberculosis bioA gene, coding for DAPA AT. Reversal of the antibiotic effect was observed on the addition of biotin or DAPA. Thus, compound 1 specifically targets DAPA AT in vivo.