Cobamides and Ribonucleotide Reduction

Hogenkamp, Harry P. C.; Ghambeer, R. K.; Brownson, Carol A.; Blakley, Raymond L.; Vitols, E.

doi:10.1016/s0021-9258(19)81736-2

Cited by 87 publications

(31 citation statements)

References 33 publications

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“…Possibility of Tritium Transfer to a Protein-Based Intermediate. Tritium transfer to a labile site on the protein has been demonstrated for AdoCbl-dependent ribonucleotide reductase (Hogenkamp et al, 1968) and ethanolamine ammonia-lyase (O'Brien et al, 1985). It was therefore of interest to determine whether tritium is transferred to the protein during the course of the glutamate mutase reaction.…”

Section: Resultsmentioning

confidence: 99%

Tritium isotope effects in adenosylcobalamin-dependent glutamate mutase: Implications for the mechanism

Marsh

1995

Biochemistry

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The transfer of tritium between adenosylcobalamin and substrate in the reaction catalyzed by glutamate mutase was examined to investigate the possibility of a protein-based radical intermediate. There was no evidence that tritium was transferred to the protein during the reaction, as tritium neither became stably bound to the protein nor exchanged with water. The kinetics of tritium transfer from adenosylcobalamin to 3-methylaspartate was investigated. Both the transfer of tritium to product and the exchange of enzyme-bound and free coenzyme contribute to the kinetics of tritium loss from adenosylcobalamin. By varying the experimental conditions, the rates of both coenzyme exchange and tritium transfer could be measured. Exchange of adenosylcobalamin with enzyme is very slow, k off = 0.01 s-1, which may reflect a conformational change in the coenzyme and/or protein involved in forming active holo enzyme. The rate constants for the loss of tritium from adenosylcobalamin and the appearance of tritium in 3-methylaspartate are much faster and very similar, k = 0.67 +/- 0.05 s-1 and k = 0.50 +/- 0.05 s-1, respectively, consistent with the transfer of tritium occurring directly between coenzyme and substrate. The isotope effect, calculated from the rate constants for tritium transfer, and kcat, determined for the overall reaction under the same conditions, are between 13.5 and 18. These values are typical of primary isotope effects seen for enzymes in which hydrogen transfer is substantially rate limiting. A protein radical, therefore, appears unlikely to feature in the mechanism of this enzyme.

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Section: Resultsmentioning

confidence: 99%

Tritium isotope effects in adenosylcobalamin-dependent glutamate mutase: Implications for the mechanism

Marsh

1995

Biochemistry

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“…The precipitate formed during evaporation was taken up in water and crystallized from water to give white crystalline [5',5'-2H2]isopropylideneadenosine (80 mg). [5',5'-2H2]-5'-Deoxyadenosylcobalamin was prepared from aquocobalamin and [5',5'-2H2]isopropylideneadenosine according to Hogenkamp et al (1968) except that the final purification was carried out by column chromatography on carboxymethylcellulose. The column (1.8 X 30 cm) was adjusted to pH 3 and washed with water before application of the sample and elution of the product with water.…”

Section: Methodsmentioning

confidence: 99%

“…In the presence of a modifier (e.g., dGTP) and a dithiol, the enzyme catalyzes rapid hydrogen exchange between water and both protons of the methylene group attached to cobalt in the coenzyme (5'-deoxyadenosylcobalamin) (Hogenkamp et al, 1968) . In the same system, but much more slowly (over 1 t From the Department of Biochemistry, College of Medicine, University of Iowa, Iowa City, Iowa, and from the Department of Biochemistry, John Curtin School of Medical Research, Australian National University, Canberra, Australia.…”

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confidence: 99%

“…This cobamide is considered to be an intermediate in both the hydrogen-exchange reaction and ribonucleotide reduction and to slowly decompose with the production of cob(II)alamin and 5 '-deoxyadenosine. The intermediate has been represented as closely related to, or identical with, cob(I)alamin plus 5'-deoxyadenosine (Hogenkamp et al, 1968) but all attempts to provide direct evidence for formation of these species have given negative results (Yamada et al, 1971).…”

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confidence: 99%

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Cobamides and ribonucleotide reduction. X. Electron paramagnetic resonance studies on cobalamin-dependent ribonucleotide reduction

Hamilton¹,

Tamao²,

Blakley³

et al. 1972

Biochemistry

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When ribonucleotide reductase from Lactobacillus leichmannii is incubated with its coenzyme (5 '-deoxyadenosylcobalamin), a thiol such as glutathione and certain nucleoside triphosphates, formation of paramagnetic products can be Dr. Lester J. Reed. The selenolipoate was reduced in the same manner as lipoate (Gunsalus and Razzell, 1957). The arabino analog of ATP (AraATP) and 2'-0-MeATP were synthesized 4696 BIOCHEMISTRY, VOL.

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“…By such processing, the total yield of 2 was 43.77 g (68%), homogeneous by TLC and NMR. An analytical sample was crystallized from ethyl acetate: Rf 0.60 (solvent A); mp 149-151°d ec; [a]23D -22°(c 1, methanol); Xma,Me0H 236 nm (t 24,300), 257.5 (17,100); XmaxKBr 3360, 1644, 1606, 1516, 1257, and 1039 cm-1; NMR data (acetone-de, 10% D20) 3.34, 3.52 (2, AB of ABX, H-5',5'a, J5s-a = 16.0 Hz, J4,b-= 6.1 Hz, J4',s'a = 4.0 Hz), 3.76 (6, s, OCH3), 4.16 (1, m, X of ABX, width 14 Hz, H-4'), 4.33 (1, t, J2-3• = =/3-4-= 3.6 Hz, H-3'), 4.40 (1, t, H-2'), 6.47 (1, d, Jr,2• = 4.1Hz, H-l'j, 6.80-6.89, 7.21-7.55 (13, m, aryl-H), and 8.18 ppm (2, s, H-2, H-8).…”

Section: Methodsmentioning

confidence: 99%

Synthesis of tritium- and deuterium-labeled 9-.beta.-D-arabinofuranosyladenine and the tritium-labeled 5'-monophosphate ester with increased metabolic stability

Baker¹,

Haskell²

1975

J. Med. Chem.

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Preparation of both a 5'-deuterium and a 5'-tritium-labeled 9-beta-D-arabinofuranosyladenine (6a and 6b) by reduction of the protected 5'-aldehyde 4 is described. Conversion of 6b to the 5'-tritium-labeled 5'-monophosphate 7b was effected directly with a phosphoryl chloride-formic acid reagent. The product 7b exhibited consistently higher blood levels of nonvolatile tritium than the 2-labeled compound when tested in dogs.

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Cobamides and Ribonucleotide Reduction

Cited by 87 publications

References 33 publications

Tritium isotope effects in adenosylcobalamin-dependent glutamate mutase: Implications for the mechanism

Tritium isotope effects in adenosylcobalamin-dependent glutamate mutase: Implications for the mechanism

Cobamides and ribonucleotide reduction. X. Electron paramagnetic resonance studies on cobalamin-dependent ribonucleotide reduction

Synthesis of tritium- and deuterium-labeled 9-.beta.-D-arabinofuranosyladenine and the tritium-labeled 5'-monophosphate ester with increased metabolic stability

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