Robert T. Taylor

Escherichia coli B N5-methyltetrahydrofolate-homocysteine vitamin-B12 transmethylase: Formation and photolability of a methylcobalamin enzyme

Abstract Extensive evidence is presented that incubation of reduced Escherichia coli 13 vitamin-B 12 transmethylase with N 5 -methyl- 14 C-tetrahydrofolate (plus unlabeled S -adenosylmethionine), with methyl- 14 C- S -adenosyImethionine alone, or with methyl- 14 C iodide resulted in the formation of a methyl- 11 C -cobalamin enzyme. Under optimal conditions N 5 -methyl- 14 C -tetrahydrofolate and methyl- 14 C-S-adenosylmethionirie yielded 0.5 equivalent and 1 equivalent, respectively, of melhyl- 14 C-cobalamin enzyme. Unlabeled N 5 -methyletrahydrofolate decreased methyl- 14 C-B 12 enzyme formation with methyl- 14 C- S -adenosylmethionine by 10-fold. Methyl- 14 C-cobalamin enzyme formation was accompanied by changes in the absorption spectrum of the initial transmethylase and produced a spectrum that displayed the characteristic of free methylcobalamin. Upon the addition of homocysteine to a methyl- 14 C-cobalamin enzyme, methionine- 14 CH 3 was obtained and the spectrum reverted back to one which was similar to that for the original enzyme. Regardless of the methyl- 14 C group donor, the methyl- 14 C-cobalamin enzyme which formed was stable to light at 0 ° unless the protein solution was acidified to pH 2.

Eschericha coli B N5-methyltetrahydrofolate-homocysteine methyl-transferase: Sequential formation of bound methylcobalamin with S-adenosyl-l-methionine and N5-methyltetrahydrofolate

Abstract The reaction parameters for methyl- 14 C-cobalamin enzyme formation with methyl- 14 C-S-adenosyl- l -methionine (methyl- 14 C-AMe) were determined and the relationship of this methylation reaction to the AMe-dependent formation of a methyl- 14 C-cobalamin enzyme with N 5 -methyl- 14 C-tetrahydrofolate ( N 5 -methyl- 14 C-H 4 -folate) was studied. Incubation of cobalamin methyltransferase either at 37 ° with methyl- 14 C-AMe alone or at 0 ° with methyl- 14 C-AMe plus unlabeled N 5 -methyl-H 4 -folate yielded about 1 equivalent of bound methyl- 14 C-cobalamin per equivalent of enzyme-bound cobalamin. A flavin reducing system was essential for methylation at both temperatures. The 37 ° methylation by methyl- 14 C-AMe was essentially complete after 1 min; whereas, the 0 ° methylation required 15–20 min and was negligible during the first 3 min. As expected, the yield of methyl- 14 C-cobalamin enzyme decreased markedly when the 0 ° methylation mixture was incubated at 37 ° because at the higher temperature the 0 ° system was converted to the corresponding system which has been employed routinely for the AMe-dependent methylation by N 5 -methyl- 14 C-H 4 -folate. Time studies at 37 ° throughout 6 min of incubation revealed that the cobalamin enzyme was first methylated by AMe within 60 sec and then methylated by N 5 -methyl-H 4 -folate over the next 6 min. A methyl- 14 C-cobalamin enzyme which had been prepared initially in a flavin reducing system was found to exchange its methyl- 14 C group with the methyl group of unlabeled N 5 -methyl-H 4 -folate. This exchange occurred aerobically and yielded an unlabeled methylcobalamin enzyme plus enzymatically active N 5 -methyl- 14 C-H 4 -folate. Alternatively, a methyl- 14 C enzyme could transfer its methyl group to H 4 -folate yielding the active isomer of N 5 -methyl- 14 C-H 4 -folate. With the use of three differently labeled 14 carbon AMes, several equivalents of this cofactor per equivalent of bound cobalamin were observed to bind tightly and non-covalently to protein in the enzyme preparations. A flavin reducing system was not required for the binding and this noncovalently bound AMe was not essential for methylation with N 5 -methyl- 14 C-H 4 -folate. Methyl iodide could be substituted for AMe and permitted methylation of the cobalamin enzyme with N 5 -methyl- 14 C-H 4 -folate. Formation of a methyl- 14 C-cobalamin enzyme with N 5 -methyl- 14 C-H 4 -folate (triglutamate) also required AMe and reduced flavin. Cobalamin enzyme preparations which were only partially dependent on AMe for methylation by N 5 -methyl- 14 C-H 4 -folate became completely dependent on AMe if they were first incubated with homocysteine in the presence of reduced flavin and dithiothreitol and then reisolated. Quantitatively, the amount of H 4 -folate which was formed upon methylation of the cobalamin enzyme with N 5 -methyl- 14 C-H 4 -folate was much less than the amount of methyl- 14 C-cobalamin enzyme which accumulated in the same incubation mixtures. Collectively, the results indicate that in our reaction system methylation of the bound cobalamin by AMe is a prerequisite to its methylation by N 5 -methyl- 14 C-H 4 -folate. Methylation by the latter is mediated by a methyl group exchange in which traces of H 4 -folate could function catalytically.

Escherichia coli B N5-methyltetrahydrofolate-homocysteine cobalamin methyltransferase: Activation with S-adenosyl-l-methionine and the mechanism for methyl group transfer

Abstract E. coli B cobalamin methyltransferase was activated upon treatment with S-adenosyl- l -methionine (AMe). Activation was attributed for several reasons to the conversion of the original cobalamin enzyme to a methylcobalamin enzyme. It resulted only when the cobalamin enzyme was incubated with AMe under the exact same reducing conditions which yield a methyl-14C-cobalamin enzyme with methyl-14C-AMe and it was prevented by propylation of the cobalamin prosthetic group. Activation was also achieved with methyl iodide. Furthermore, the ability of an activated cobalamin enzyme to synthesize methionine aerobically was lost if it was exposed to either tetrahydrofolate or homocysteine at 37 ° prior to an incubation with N5-methyl-14C-tetrahydrofolate (N5-methyl-14C-H4-folate) plus homocysteine. This inactivation was correlated with the demethylation of a methyl-14C-cobalamin enzyme which had been prepared with methyl-14C-AMe. A methylcobalamin enzyme, freed of unbound AMe, catalyzed limited N5-methyl-H4-folate-homocysteine transmethylation in the absence of reduced flavin. Based on the total amount of bound cobalamin (ca. 0.4 mμmole) a minimum turnover of 8–10 fold was observed aerobically. Methionine synthesis was limited under aerobic conditions by the rapid inactivation of the cobalamin enzyme during catalysis. The total turnover by 0.05–0.1 mμmole of methylcobalamin enzyme was increased to about 100-fold when the reaction mixtures were incubated either in a vacuum or else under nitrogen gas. Exogenous AMe did not improve the amount of catalysis by a methylcobalamin enzyme when reduced flavin was absent. The original, unmethylated cobalamin enzyme did not display the limited catalytic activity which was observed with a methylcobalamin enzyme. In a flavin-reducing system AMe was essential for 1.0 μμmole of both the methyl cobalamin enzyme and the original cobalamin enzyme to maintain a constant rate of N5-methyl-H4-folate-homocysteine transmethylation. When 1.8 mμmoles of unlabeled methylcobalamin enzyme were incubated with an excess of N5-methyl-14C-H4-folate and 0.1–0.4 mμmole of homocysteine (added simultaneously) the resulting methyl-14C-methionine had a much lower specific activity than the substrate methyl-14C-donor. The synthesized methyl-14C-methionine had the same specific activity, however, if the unlabeled methylcobalamin enzyme was incubated first with N5-methyl-14C-H4-folate and then with 0.1–0.4 mμmole of homocysteine. By this prior treatment with N5-methyl-14C-H4-folate, methyl-14C groups were exchanged onto the cobalt atom without appreciably inactivating the enzyme for subsequent aerobic methyl-14C-methionine synthesis. Additional catalytic properties of the cobalamin methyltransferase were studied. The enzyme was observed to catalyze a methyl group exchange reaction between pteridine ring labeled N5-methyl-H4-folate and unlabeled H4-folate. Exchange trans-methylation required AMe as a cofactor but was not dependent upon reduced flavin because the high concentration of H4-folate which was employed as a substrate also apparently functioned as a reducing agent for the cobalamin enzyme. This conclusion was based upon the finding that H4-folate can be substituted for reduced flavin and permit methyl-14C-methionine synthesis from N5-methyl-14C-H4-folate plus homocysteine at 1 3 the rate attainable in the presence of reduced flavin. Catalysis of methyl group transfer from AMe to H4-folate was demonstrated and in this reaction too, reduced flavin was not essential. H4-Folate (1.5 m m ) inhibited by 2.4-fold the rate of N5-methyl-H4-folate-homocysteine transmethylation in the presence of reduced flavin; however, it increased by 2.4–3.0-fold the rate of AMe-homocysteine transmethylation in the presence of reduced flavin. Chemical propylation of the cobalamin prosthetic group inhibited equally and in a light reversible manner methyl group transfer from AMe to H4-folate; from N5-methyl-H4-folate to homocysteine; and from AMe to homocysteine (± H4-folate). Additional data were also collected on the formation and breakdown of a methyl-14C-eobalamin enzyme. Within the first minute of incubation at 37 ° the rate of AMe-dependent methyl-14C-cobalamin enzyme formation from N5-methyl-14C-H4-folate was increased by 4–5-fold when limiting amounts of either homocysteine or H4-folate were added to the system. Contrary to what was previously observed with a 1:1 ratio of active N5-methyl-14C-H4-folate to cobalamin enzyme, dithiothreitol was not required in order to obtain maximal amounts of methyl-14C-cobalamin enzyme when a 10:1 ratio was used. Nevertheless, the rate of methyl-14C-cobalamin enzyme formation was slower in the absence of dithiothreitol, unless limiting quantities of homocysteine were present. Demethylation of a methyl-14C-cobalamin enzyme with homocysteine was biphasic. It consisted of an initial rapid reaction which was complete in less than 5 sec at 37 ° and a very slow reaction which spanned the next 15 min at 37 °. About 70% of the methyl-14C-groups were transferred to homocysteine within the first 5 sec at a rate which was too rapid to measure. On the basis of the foregoing results and other recent work, a reaction mechanism for N5-methyl-H4-folate-homocysteine transmethylation is suggested and discussed in detail. In the reaction scheme which is proposed methyl groups from N5-methyl-H4-folate are transferred to homocysteine via a methylcobalamin enzyme intermediate. AMe is assigned the possible role of an “activator” which methylates only inactive forms of the cobalamin protein, as, for example, the cobalamin enzyme species which one isolates from cell-free extracts.

The role of vincristine in the treatment of childhood acute leukemia

In a study of 117 patients under the age of 20 with acute leukemia, vincristine (VCR), at 2 mg. per square meter body surface per week, produced complete remissions in 55 per cent and partial remissions in 15 per cent. The drug also induced second remissions. Patients entering complete remission with VCR were randomly allocated to maintenance therapy with VCR or placebo. The median duration of remission was short: 9 weeks for VCR compared with 6 weeks for placebo. The probability of serious neurological toxicity computed according to the time of exposure to VCR, based on the supposition that VCR was not used for maintenance therapy, indicated that the minimal theoretical risk of toxicity for the highest complete remission rate occurred at 4 weeks (38 per cent remissions with 5 per cent toxicity). At 6 weeks, the corresponding probabilities were 54 and 16 per cent.

Studies on the nature of the bound cobamide in E. coli N5-methyltetrahydrofolate-homocysteine transmethylase

Abstract N 5 -methyl-H 4 -folate-homocysteine transmethylase, purified 100-fold from E. coli , was studied with respect to the content and the chemical nature of its cobamide prosthetic group. Extraction of the enzyme with hot ethanol in the absence of alkaline cyanide removed 50% of the bound eobamide. A light-sensitive cobamide containing 5,6-dimethylbenzimidazole and exhibiting the spectrophotometric, Chromatographic, and electrophoretic characteristics of sulphito-B 12 was obtained. In addition, various cobamides protected the apoenzyme form of the enzyme from inactivation by parachloromercuribenzoate.

Enzymic synthesis of methionine: Formation of a radioactive cobamide enzyme with N5-methyl-14C-tetrahydrofolate

Abstract When extensively purified preparations of N 5 -methyltetrahydrofolate-homocysteine transmethylase were incubated with N 5 -methyl- 14 C-tetrahydrofolate in the presence of S -adenosyl- l -methionine and a FMNH 2 -dithiothreitol reducing system, 14 C-labeled protein was obtained. This radioactivity was stable to light and the amount corresponded to at least 0.3 mμmole of bound 14 C per mμmole of B 12 -chromophore. Upon incubation of the 14 C-labeled protein with excess homocysteine, methionine- 14 CH 3 was formed in nearly stoichiometric quantities. Propylation of the B 12 -chromophore on the transmethylase inhibited the rate of catalysis of methionine synthesis and the amount of 14 C labeling from N 5 -methyl- 14 C-tetrahydrofolate to the same extent. All the available evidence indicates that a cobamide enzyme containing methyl- 14 C groups was isolated.

Control of one-carbon metabolism in a methionine-B12 auxotroph of Escherichia coli

Abstract The control mechanisms of N 5,10 -methylene-H 4 -folate metabolism and de novo methyl group synthesis were studied in a mutant Escherichia coli K 12 , which is auxotrophic for either l -methionine or vitamin-B 12 . N 5,10 -Methylene-H 4 -folate reductase was found to be repressed at high media concentrations of l -methionine. Growth at high media concentrations of purines led to a partial repression of N 5,10 -methylene-H 4 -folate dehydrogenase. This enzyme was also found to be inhibited in vitro by purine ribonucleotide triphosphates, and the kinetics of the inhibition indicated that they were competitive with triphosphopyridine nucleotide. Tracer studies of the incorporation of 3- 14 C- l -serine into nucleic acid, protein, and phospholipid correlated with the preceding in vivo and in vitro effects.

Chemical propylation of vitamin-B12 transmethylase: Anomalous behavior of S-adenosyl-l-methionine

Abstract The requirement for S -adenosyl- l -methionine (AMe) in the chemical propylalation of Escherichia coli N 5 -methyltetrahydrofolate-homocysteine transmethylase has been reexamined. When a relatively crude ammonium sulfate fraction was used as a source of the enzyme, AMe was an essential requirement for propylation in a flavin reducing system; however, exogenous AMe exhibited only a stimulatory effect in propylation systems which also contained dithiothreitol. With more purified transmethylase preparations, AMe inhibited chemical propylation in a dithiothreitol-containing incubation system. Evidence is presented to substantiate that AMe inhibits chemical propylation by initially methylating the bound cobalamin. This inhibition by AMe was completely reversed by only two cobalamin-enzyme equivalents of homocysteine. The inability of AMe to inhibit chemical propylation in the unpurified enzyme has been attributed to the degradation of AMe preparations to homocysteine by contaminating proteins in the crude enzyme preparation. The formation of small amounts of homocysteine ( N 5 -methyL- 14 C-tetrahydrofolate. A possible scheme to accommodate the previous [ J. Biol. Chem. 240 , 3064 (1965)] apparent requirement for AMe in propylation is suggested. Coincidental in this reinvestigation of the requirement for AMe in propylation was the finding that crystalline preparations of bovine serum albumin contained trace amounts of homocysteine which appeared to be bound through a disulfide bond.

[110] Branched-chain amino acid aminotransferase (pig heart, soluble)

Publisher Summary This chapter presents the assay, purification, and properties of aminotransferase from pig heart. When leucine is used as the substrate, the 2, 4-dinitrophenylhydrazone of α- ketoisocaproate may be selectively extracted by cyclo hexane from acidic solutions containing an excess of α -ketoglutarate 2, 4-dinitrophenylhydrazone. The enzyme is sensitive to sulfhydryl reagents. The loss of activity upon storage at high pH values may be recovered by treatment with thiols. Just as other transaminases, the enzyme is inhibited by carboxylic acid substrate analogs and by a variety of carbonyl reagents, especially phenylhydrazine and hydroxylamine. The calculated sedimentation constant for zero protein concentration is 5.1 S. With 6.7 m M substrate concentrations, the assay shows a sharp optimum at about pH 8.3. Further analysis of this pH activity curve shows that the decrease on the acid side of the optimum is due to both a decrease in maximum velocity and an increase in the K m for leucine. The decrease on the basic side of the optimum, which is due to an increase in the K m for ketoglutarate, does not occur when transamination from leucine to α -ketoisovalerate is studied.

US flu mortality estimates are based on solid science

Editor—Doshi implies that the use of statistical models to estimate flu related mortality is inappropriate.1 Not so. Epidemiologists rely on statistical models because the International Classification of Diseases (ICD) code for influenza (ICD9 487) severely undercounts the true number of flu related deaths. Doshi also implies that the Centers for Disease Control and Prevention (CDC) deliberately exaggerates flu mortality for the benefit of the pharmaceutical industry, while other scientists stand by, meek and mute. The BMJ s decision to publish Doshis commentary with no counterpoint from …