Recombinant human DNA (cytosine-5) methyltransferase. II. Steady-state kinetics reveal allosteric activation by methylated dna.

Initial velocity determinations were conducted with human DNA (cytosine-5) methyltransferase (DNMT1) on unmethylated and hemimethylated DNA templates in order to assess the mechanism of the reaction. Initial velocity data with DNA and S-adenosylmethionine (AdoMet) as variable substrates and product inhibition studies with methylated DNA and S-adenosylhomocysteine (AdoHcy) were obtained and evaluated as double-reciprocal plots. These relationships were linear for plasmid DNA, exon-1 from the imprinted small nuclear ribonucleoprotein-associated polypeptide N, (CGG.CCG)(12), (m(5)CGG. CCG)(12), and (CGG.CCG)(73) but were not linear for (CGG. Cm(5)CG)(12). Inhibition by AdoHcy was apparently competitive versus AdoMet and uncompetitive/noncompetitive versus DNA at 相似文献   

Phosphatidylethanolamine N-methyltransferase in human red blood cell membrane preparations. Kinetic mechanism

The successive methylations of phosphatidylethanolamine to form phosphatidylcholine were measured using exogenously added intermediates and membrane preparations from human red blood cells. The addition of phosphatidylethanolamine resulted in no increase in methylation rate over that with endogenous substrate; however, the addition of monomethylphosphatidylethanolamine (PME) and dimethylphosphatidylethanolamine (PDE) markedly increased the reaction rate and allowed studies into the kinetic mechanism for the second and third methylation reactions. The data are consistent with catalysis of the last two methylations being by a single enzyme with a random Bi-Bi sequential mechanism. Analysis of PDE:phosphatidylcholine product ratios indicates that the enzyme can conduct multiple methylations of enzyme-bound phospholipid. The nature of the acyl chain (16:0 versus 18:1) of the phospholipid had only a small effect on the value of the kinetic constants. The maximal velocities obtained with the 18:1 substrate were less than 5% lower than those obtained with the 16:0 substrate. The Km values for the two phospholipids were 20-45 and 10-14 microM for the methylation of PME and PDE, respectively. The Km for S-adenosylmethionine (AdoMet) was 5-9 microM with PME and 4 microM with PDE as substrates. Depending on the acyl chain and the phospholipid, the Ki(AdoMet) varied from 8 to 19 microM, the Ki(PME) from 41 to 82 microM, and the Ki(PDE) from 35 to 61 microM. The Ki for S-adenosylhomocysteine (AdoHcy) was between 1.0 and 1.4 microM depending upon the variable substrate. The endogenous concentrations of PME and PDE in red blood cell membranes were estimated to be 0.49 and 0.24 mumol/liter packed cells, respectively. The product from the utilization of AdoMet, S-adenosylhomocysteine (AdoHcy), was shown to be a competitive inhibitor of its precursor, AdoMet, and a noncompetitive inhibitor of the two phospholipid substrates.     

Kinetic mechanism of the tRNA-modifying enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA)

The bacterial enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) catalyzes the unprecedented transfer and isomerization of the ribosyl moiety of S-adenosylmethionine (AdoMet) to a modified tRNA nucleoside in the biosynthesis of the hypermodified nucleoside queuosine. The complexity of this reaction makes it a compelling problem in fundamental mechanistic enzymology, and as part of our mechanistic studies of the QueA-catalyzed reaction, we report here the elucidation of the steady-state kinetic mechanism. Bi-substrate kinetic analysis gave initial velocity patterns indicating a sequential mechanism, and provided the following kinetic constants: K (M)(tRNA)= 1.9 +/- 0.7 microM and K (M)(AdoMet)= 98 +/- 5.0 microM. Dead-end inhibition studies with the substrate analogues S-adenosylhomocysteine and sinefungin gave competitive inhibition patterns against AdoMet and noncompetitive patterns against preQ(1)-tRNA(Tyr), with K(i) values of 133 +/- 18 and 4.6 +/- 0.5 microM for sinefungin and S-adenosylhomocysteine, respectively. Product inhibition by adenine was noncompetitive against both substrates under conditions with a subsaturating cosubstrate concentration and uncompetitive against preQ(1)-tRNA(Tyr) when AdoMet was saturating. Inhibition by the tRNA product (oQ-tRNA(Tyr)) was competitive and noncompetitive against the substrates preQ(1)-tRNA(Tyr) and AdoMet, respectively. Inhibition by methionine was uncompetitive versus preQ(1)-tRNA(Tyr), but noncompetitive against AdoMet. However, when methionine inhibition was investigated at high AdoMet concentrations, the pattern was uncompetitive. Taken together, the data are consistent with a fully ordered sequential bi-ter kinetic mechanism in which preQ(1)-tRNA(Tyr) binds first followed by AdoMet, with product release in the order adenine, methionine, and oQ-tRNA. The chemical mechanism that we previously proposed for the QueA-catalyzed reaction [Daoud Kinzie, S., Thern, B., and Iwata-Reuyl, D. (2000) Org. Lett. 2, 1307-1310] is consistent with the constraints imposed by the kinetic mechanism determined here, and we suggest that the magnitude of the inhibition constants for the dead-end inhibitors may provide insight into the catalytic strategy employed by the enzyme.     

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.     

Kinetic mechanism of isoprenylated protein methyltransferase.

The kinetic mechanism of the rod outer segment (ROS) isoprenylated protein methyltransferase was investigated. This S-adenosyl-L-methionine (AdoMet)-linked enzyme transfers methyl groups to carboxyl-terminal isoprenylated cysteine residues of proteins, generating methyl esters. The enzyme also processes simple substrates such as N-acetyl-S-farnesyl-L-cysteine (L-AFC). Initial studies showed that a ping-pong Bi Bi mechanism could be eliminated. In a ping-pong Bi Bi mechanism plots of 1/v versus 1/[substrate A] at different fixed substrate B concentrations are expected to yield a family of parallel lines whose slopes equal Km/Vmax. In fact, converging curves were found, which suggested a sequential mechanism. Dead-end inhibitors were used in order to further investigate the kinetic mechanism. S-Farnesylthioacetic acid is shown to be a dead-end competitive inhibitor with respect to the prenylated substrate L-AFC. On the other hand, S-farnesylthioacetic acid proved to be uncompetitive with respect to AdoMet, suggesting an ordered mechanism with AdoMet binding first. Further evidence for this mechanism came from product inhibition studies using the methyl ester of L-AFC (L-AFCMe) and S-adenosyl-L-homocysteine (AdoHcy). Since AdoMet binds first to the enzyme, one of the products (L-AFCMe or AdoHcy) should be a competitive inhibitor with respect to it. It could be shown that AdoHcy is a competitive inhibitor with respect to AdoMet, but L-AFCMe is a mixed-type inhibitor both with respect to AdoMet and to L-AFC. Therefore, AdoHcy combines with the same enzyme form as does AdoMet, and must be released from the enzyme last. Moreover, L-AFC and L-AFCMe must bind to different forms of the enzyme.     

The effect of S-adenosyl methionine on the TSH receptor function in human thyroid tissue: increase in binding of TSH and decrease in adenylate cyclase coupling

To determine the effects of S-Adenosyl methionine (AdoMet) on TSH receptor function and adenylate cyclase coupling, human thyroidal crude membrane fraction was pretreated with AdoMet and with S-Adenosyl homocysteine (AdoHcy), separately or in combination. 125I-TSH binding to the pretreated membrane and adenylate cyclase activity of the membrane were examined. In contrast to the reported effect of AdoMet on the decrease in GH binding to lactogenic receptor, AdoMet 0.5 mumoles/ml significantly increased the binding of TSH to the receptor by increasing the affinity of the binding, whereas it decreased the coupling of adenylate cyclase significantly. The effect of AdoMet was partially counteracted by the pretreatment of the membrane with AdoHcy. This effect of AdoMet is very similar to that of diamide previously reported. The result implies that the effect is due to an alteration in the tertiary structure of receptor protein triggered by methylation.     

Methyltransferase CheR binds to its chemoreceptor substrates independent of their signaling conformation yet modifies them differentially

Methylation of specific chemoreceptor glutamyl residues by methyltransferase CheR mediates sensory adaptation and gradient sensing in bacterial chemotaxis. Enzyme action is a function of chemoreceptor signaling conformation: kinase‐off receptors are more readily methylated than kinase‐on, a feature central to adaptational and gradient‐sensing mechanisms. Differential enzyme action could reflect differential binding, catalysis or both. We investigated by measuring CheR binding to kinase‐off and kinase‐on forms of Escherichia coli aspartate receptor Tar deleted of its CheR‐tethering, carboxyl terminus pentapeptide. This allowed characterization of the low‐affinity binding of enzyme to the substrate receptor body, otherwise masked by high‐affinity interaction with pentapeptide. We quantified the low‐affinity protein–protein interactions by determining kinetic rate constants of association and dissociation using bio‐layer interferometry and from those values calculating equilibrium constants. Whether Tar signaling conformations were shifted by ligand occupancy or adaptational modification, there was little or no difference between the two signaling conformations in kinetic or equilibrium parameters of enzyme‐receptor binding. Thus, differential methyltransferase action does not reflect differential binding. Instead, the predominant determinants of binding must be common to different signaling conformations. Characterization of the dependence of association rate constants on Deybe length, a measure of the influence of electrostatics, implicated electrostatic interactions as a common binding determinant. Taken together, our observations indicate that differential action of methyltransferase on kinase‐off and kinase‐on chemoreceptors is not the result of differential binding and suggest it reflects differential catalytic propensity. Differential catalysis rather than binding could well be central to other enzymes distinguishing alternative conformations of protein substrates.     

Biosynthetic direction substrate kinetics and product inhibition studies on the first enzyme of histidine biosynthesis, adenosine triphosphate phosphoribosyltransferase.

Initial velocity steady-state substrate kinetics for the ATP phosphoribosyltransferase reaction in the biosynthetic direction were determined and are consistent with a sequential kinetic mechanism. To hold the fractions of magnesium-complexed substrates and products constant so as to avoid possible distortion of reciprocal velocity plots Mg²⁺ binding constants to the substrates ATP and phosphoribosylpyrophosphate and the product pyrophosphate were measured under assay conditions. Several conformational states of the phosphoribosyltransferase distinguishable by other criteria gave similar substrate kinetic behavior. Product inhibition studies were conducted to elucidate the binding order. Phosphoribosyl-ATP was competitive with respect to ATP and was non-competitive with respect to phosphoribosylpyrophosphate. Pyrophosphate was non-competitive with respect to both substrates. The data are consistent with the ordered Bi-Bi kinetic mechanism with ATP binding first to free enzyme and phosphoribosyl-ATP dissociating last from enzyme-product complexes.     

Human Brain 6-Phosphogluconate Dehydrogenase: Purification and Kinetic Properties

6-Phosphogluconate dehydrogenase has been purified from human brain to a specific activity of 22.8 U/mg protein. The molecular weight was 90,000. At low ionic strengths enzyme activity increased, due to an increase in Vmax and a decrease in Km for 6-phosphogluconate, and activity subsequently decreased as the ionic strength was increased (above 0.12). Both 6-phosphogluconate and NADP+ provided good protection against thermal inactivation, with 6-phosphogluconate also providing considerable protection against loss of activity caused by p-chloromercuribenzoate and iodoacetamide. Initial velocity studies indicated the enzyme mechanism was sequential. NADPH was a competitive inhibitor with respect to NADP+, and the Ki values for this inhibition were dependent on the concentration of 6-phosphogluconate. Product inhibition by NADPH was noncompetitive when 6-phosphogluconate was the variable substrate, whereas inhibition by the products CO2 and ribulose 5-phosphogluconate and NADP+ were varied. In totality these data suggest that binding of substrates to the enzyme is random. CO2 and ribulose 5-phosphate are released from the enzyme in random order with NADPH as the last product released.     

Direct photolabeling of the EcoRII methyltransferase with S-adenosyl-L-methionine

Ultraviolet irradiation of EcoRII methyltransferase in the presence of its substrate, S-adenosyl-L-methionine (AdoMet), results in the formation of a stable enzyme-substrate adduct. This adduct can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after irradiation of the enzyme in the presence of either [methyl-3H]AdoMet or [35S]AdoMet. The extent of photolabeling is low. Under optimal conditions, 4.5 pmol of [3H]AdoMet is incorporated into 100 pmol of enzyme. Use of the 8-azido derivative of AdoMet as the photolabeling substrate increases the incorporation by approximately 2-fold. However, this adduct, unlike the one formed with AdoMet, is not stable when treated with thiol reagents or precipitated with trichloroacetic acid. A catalytically active conformation of the enzyme is needed for AdoMet photolabeling. Heat-inactivated enzyme or proteins for which AdoMet is not a substrate or cofactor do not undergo adduct formation. Two other methyltransferases, MspI and dam methylases are also shown to form adducts with AdoMet upon UV irradiation. The binding constant of the EcoRII methyltransferase for AdoMet determined with the photolabeling reaction is 11 microM, which is similar to the binding constant of 9 microM previously reported (Friedman, S. (1986) Nucleic Acids Res. 14, 4543-4556). The AdoMet analogs S-adenosyl-L-homocysteine (Ki = 0.83 microM) and sinefungin (Ki = 4.3 microM) are effective inhibitors of photolabeling, whereas S-adenosyl-D-homocysteine (Ki = 46 microM) is a poor inhibitor. These experiments indicate that AdoMet becomes covalently bound at the AdoMet-binding site on the enzyme molecule. The EcoRII methyltransferase-AdoMet adduct is very stable and could be used to identify the AdoMet-binding site on DNA methyltransferases.     

The role of glutamate 87 in the kinetic mechanism of Thermus thermophilus isopropylmalate dehydrogenase.

The kinetic mechanism of the oxidative decarboxylation of 2R,3S-isopropylmalate by the NAD-dependent isopropylmalate dehydrogenase of Thermus thermophilus was investigated. Initial rate results typical of random or steady-state ordered sequential mechanisms are obtained for both the wild-type and two mutant enzymes (E87G and E87Q) regardless of whether natural or alternative substrates (2R-malate, 2R,3S-tartrate and/or NADP) are utilized. Initial rate data fail to converge on a rapid equilibrium-ordered pattern despite marked reductions in specificity (kcat/Km) caused by the mutations and alternative substrates. Although the inhibition studies alone might suggest an ordered kinetic mechanism with cofactor binding first, a detailed analysis reveals that the expected noncompetitive patterns appear uncompetitive because the dissociation constants from the ternary complexes are far smaller than those from the binary complexes. Equilibrium fluorescence studies both confirm the random binding of substrates and the kinetic estimates of the dissociation constants of the substrates from the binary complexes. The latter are not distributed markedly by the mutations at site 87. Mutations at site 87 do not affect the dissociation constants from the binary complexes, but do greatly increase the Michaelis constants, indicating that E87 helps stabilize the Michaelis complex of the wild-type enzyme. The available structural data, the patterns of the kinetics results, and the structure of a pseudo-Michaelis complex of the homologous isocitrate dehydrogenase of Escherichia coli suggest that E87 interacts with the nicotinamide ring.     

Purification and properties of a NAD(P)H:flavin oxidoreductase from the luminous bacterium, Beneckea harveyi.

A NAD(P)H:flavin oxidoreductase, which produces FMNH2, one of the substrates for the luciferase reaction in bioluminescent bacteria, has been purified with the aid of affinity chromatography on epsilon-aminohexanoyl-FMN-Sepharose. The purified enzyme, isolated from Beneckea harveyi, had a specific activity of 89 mumol of NADH oxidized/min/mg of protein at 23 degrees in the presence of saturating FMN and NADH and appeared homogeneous by several criteria on polyacrylamide gel electrophoresis. A molecular weight of 24,000 was estimated both by gel filtration and and sodium dodecyl sulfate gel electrophoresis indicating that the enzyme is composed of a single polypeptide chain. Kinetic studies showed that the higher specificity of the enzyme for NADH than NADPH and for riboflavin and FMN than FAD was primarily due to variations in the Michaelis constants for the different substrates. Initial velocity studies with all pairs of substrates gave intersecting patterns supporting a sequential mechanism for the NAD(P)H:flavin oxidoreductase.     

Assignment of enzymatic functions to specific regions of the PLP-dependent heme protein cystathionine beta-synthase.

Cystathionine beta-synthase is a unique heme protein that catalyzes a pyridoxal phosphate (or PLP)-dependent beta-replacement reaction. The reaction involves the condensation of serine and homocysteine and constitutes one of the two major avenues for detoxification of homocysteine in mammals. The enzyme is allosterically regulated by S-adenosylmethionine (AdoMet). In this study, we have characterized the kinetic, spectroscopic, and ligand binding properties of a truncated catalytic core of cystathionine beta-synthase extending from residues 1 through 408 in which the C-terminal 143 residues have been deleted. This is similar to a natural variant of the protein that has been described in a homocystinuric patient in which the predicted peptide is 419 amino acids in length. Truncation leads to the formation of a dimeric enzyme in contrast to the tetrameric organization of the native enzyme. Some of the kinetic properties of the truncated enzyme are different from the full-length form, most notably, significantly higher K(m)s for the two substrates, and loss of activation by AdoMet. This is paralleled by the absence of AdoMet binding to the truncated form, whereas four AdoMet molecules bind cooperatively to the full-length tetrameric enzyme with a K(d) of 7. 4 microM. Steady-state kinetic analysis indicates that the order of substrate addition is important. Thus, preincubation of the enzyme with homocysteine leads to a 2-fold increase in V(max) relative to preincubation of the enzyme with serine. Since the intracellular concentration of serine is significantly greater than that of homocysteine, the physiological significance of this phenomenon needs to be considered. Based on ligand binding studies and homology searches with protein sequences in the database, we assign residues 68-209 as being important for PLP binding, residues 241-341 for heme binding, and residues 421-469 for AdoMet binding.     

Kinetic studies of human liver ferrochelatase. Role of endogenous metals

Ferrochelatase activity in human liver has been extensively characterized in the mitochondrial fraction by kinetic study of the enzyme in initial velocity conditions. We found that human liver mitochondrial membranes contain large amounts of endogenous metals that are substrates for the enzyme, leading to a lack of linearity of the activity as function of protein concentration. This lack of linearity is mainly due to a high zinc-chelatase activity with endogenous zinc. Under optimal experimental conditions, the maximum velocity for iron incorporation was 8.7 nmol of protoheme/h/mg of protein, and the maximum velocity for zinc incorporation was 4.3 nmol of zinc-protoporphyrin/h/mg of protein. The Michaelis constant for protoporphyrin IX was (i) dependent on the amount of protein when the overall chelatase reactions were measured but (ii) independent of the amount of protein when only zinc-chelatase activity was measured (Km = 0.5 microM). The Michaelis constants for iron and zinc were 0.35 and 0.08 microM, respectively, and the inhibitory constants for competitive incorporation of iron and zinc were KIFe/Zn = 0.12 microM and KIZn/Fe = 0.58 microM. The affinity of the enzyme for zinc lowers the actual determination of ferrochelatase activity with iron as substrate. Furthermore, when measuring ferrochelatase (e.g. in liver biopsy), endogenous zinc content in the biological sample must be taken into account.     

The cytochrome c peroxidase-catalyzed oxidation of ferrocytochrome c by hydrogen peroxide. Steady state kinetic mechanism

Initial velocities for the cytochrome c peroxidase-catalyzed oxidation of ferrocytochrome c by hydrogen peroxide have been measured as functions of both the ferrocytochrome c (0.27-104 microM) and hydrogen peroxide (0.25-200 microM) concentrations at 25 degrees C, 0.01 M ionic strength, and pH 7 in a cacodylate/KNO3 buffer system Eadie-Hofstee plots of the initial velocity as a function of ferrocytochrome c concentration at constant hydrogen peroxide are nonlinear. A mechanism is proposed which includes random addition of the two substrates to the enzyme and a single catalytically active cytochrome c binding site. The mechanism is consistent with prior studies on cytochrome c peroxidase and fits the steady state kinetic data well.     

The binding of substrates and a product of the enzymatic reaction to glutathione S-transferase A.

The binding of substrates and a product to glutathione S-transferase A from rat liver was studied by use of equilibrium dialysis and equilibrium partition in a two-phase system. The radioactive substrates glutathione and bromosulfophthalein as well as a product of glutathione and 3,4-dichloro-1-nitrobenzene, S-(2-chloro-4-nitrophenyl)glutathione, gave hyperbolic binding isotherms with a stoichiometry of 2 mol per mol of enzyme (i.e. 1 molecule per subunit). Glutathione (and glutathione disulfide) had an equilibrium (dissociation) constant for the binding of about 10 microM, whereas bromosulfophthalein and the product had equilibrium constants of about 0.5 microM. All ligands showed the same binding stoichiometry, and competition experiments involving unlabeled ligands indicated that glutathione and the glutathione derivatives were binding to the same site. Low affinity sites appeared to exist in addition to the specific high affinity sites (one per subunit) for all ligands tested. The binding studies are fully consistent with a steady state random kinetic mechanism for the enzyme.     

Mechanistic studies of the long chain acyl-CoA synthetase Faa1p from Saccharomyces cerevisiae

Long chain acyl-CoA synthetase (ACSL; fatty acid CoA ligase: AMP forming; EC 6.2.1.3) catalyzes the formation of acyl-CoA through a process, which requires fatty acid, ATP and coenzymeA as substrates. In the yeast Saccharomyces cerevisiae the principal ACSL is Faa1p (encoded by the FAA1 gene). The preferred substrates for this enzyme are cis-monounsaturated long chain fatty acids. Our previous work has shown Faa1p is a principal component of a fatty acid transport/activation complex that also includes the fatty acid transport protein Fat1p. In the present work hexameric histidine tagged Faa1p was purified to homogeneity through a two-step process in the presence of 0.1% eta-dodecyl-beta-maltoside following expression at 15 degrees C in Escherichia coli. In order to further define the role of this enzyme in fatty acid transport-coupled activation (vectorial acylation), initial velocity kinetic studies were completed to define the kinetic parameters of Faa1p in response to the different substrates and to define mechanism. These studies showed Faa1p had a Vmax of 158.2 nmol/min/mg protein and a Km of 71.1 microM oleate. When the concentration of oleate was held constant at 50 microM, the Km for CoA and ATP were 18.3 microM and 51.6 microM respectively. These initial velocity studies demonstrated the enzyme mechanism for Faa1p was Bi Uni Uni Bi Ping Pong.     

Biosynthesis of phytosterols. Kinetic mechanism for the enzymatic C-methylation of sterols

Cloned soybean sterol methyltransferase was purified from Escherichia coli to gel electrophoretic homogeneity. From initial velocity experiments, catalytic constants for substrates best suited for the first and second C1 transfer activities, cycloartenol and 24(28)-methylenelophenol, were 0.01 and 0.001 s-1, respectively. Two-substrate kinetic analysis using cycloartenol and S-adenosyl-l-methionine (AdoMet) generated an intersecting line pattern characteristic of a ternary complex kinetic mechanism. The high energy intermediate analog 25-azacycloartanol was a noncompetitive inhibitor versus cycloartenol and an uncompetitive inhibitor versus AdoMet. The dead end inhibitor analog cyclolaudenol was competitive versus cycloartenol and uncompetitive versus AdoMet. 24(28)-Methylenecycloartanol and AdoHcy generated competitive and noncompetitive kinetic patterns, respectively, with respect to AdoMet. Therefore, 24(28)-methylenecycloartanol combines with the same enzyme form as does cycloartenol and must be released from the enzyme before AdoHcy. 25-Azacycloartanol inhibited the first and second C1 transfer activities with about equal efficacy (Ki = 45 nm), suggesting that the successive C-methylation of the Delta 24 bond occurs at the same active center. Comparison of the initial velocity data using AdoMet versus [2H3-methyl]AdoMet as substrates tested against saturating amounts of cycloartenol indicated an isotope effect on VCH3/VCD3 close to unity. [25-2H]24(28)-Methylenecycloartanol, [28E-2H]24 (28)-methylenelanosterol, and [28Z-2H]24(28)-methylene lanosterol were prepared and paired with AdoMet or [methyl-3H3]AdoMet to examine the kinetic isotope effects attending the C-28 deprotonation in the enzymatic synthesis of 24-ethyl(idene) sterols. The stereochemical features as well as the observation of isotopically sensitive branching during the second C-methylation suggests that the two methylation steps can proceed by a change in chemical mechanism resulting from differences in sterol structure, concerted versus carbocation; the kinetic mechanism remains the same during the consecutive methylation of the Delta 24 bond.     

Purification and steady-state kinetic analysis of yeast thiosulfate reductase.

Thiosulfate reductase has been purified approximately 70-fold from an extract of bakers' yeast. An enzyme with a molecular weight of 17,000, a Stokes radius of 19 Å, and a pI of 5.1 was obtained. Initial velocity and inhibition studies indicate that the substrates add in a random fashion. Further evidence suggests that the rapid-equilibrium assumption is not totally applicable. The enzyme has two distinct but closely situated substrate binding sites—one for compounds with an RSO₃^? structure and one for the sulfhydryl substrate.