1995
DOI: 10.1021/bi00022a030
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Tritium isotope effects in adenosylcobalamin-dependent glutamate mutase: Implications for the mechanism

Abstract: 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 o… Show more

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Cited by 42 publications
(53 citation statements)
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“…3-Methylaspartase was purified as described by Hsiang and Bright (16). 5Ј-[ 3 H]AdoCbl was synthesized by using glutamate mutase to catalyze the exchange of tritium from tritiated glutamate to AdoCbl (9). AdoCbl and 2-KG were purchased from Sigma; tritiated glutamic acid was purchased from Amersham PLC.…”
Section: Methodsmentioning
confidence: 99%
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“…3-Methylaspartase was purified as described by Hsiang and Bright (16). 5Ј-[ 3 H]AdoCbl was synthesized by using glutamate mutase to catalyze the exchange of tritium from tritiated glutamate to AdoCbl (9). AdoCbl and 2-KG were purchased from Sigma; tritiated glutamic acid was purchased from Amersham PLC.…”
Section: Methodsmentioning
confidence: 99%
“…AdoCbl and 2-KG were purchased from Sigma; tritiated glutamic acid was purchased from Amersham PLC. The sources of other materials have been described previously (9,15) or were purchased from commercial suppliers.…”
Section: Methodsmentioning
confidence: 99%
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“…The purification from recombinant E. coli of the engineered single subunit glutamate mutase protein, GlmES, which has been used in this and previous mechanistic studies, has been described previously (17 (18). [8][9][10][11][12][13][14] C]-adenosylcobalamin was synthesized using established protocols described by Brown et.…”
Section: Experimental Procedures Materialsmentioning
confidence: 99%
“…The sources of other materials have been described previously [3,4,22,27] or were purchased from commercial suppliers. [5'-3 H]-AdoCbl was prepared enzymatically by using GlmES protein to exchange tritium into the coenzyme from 3 H-labelled glutamate in a slight modification of the procedure described previously [31]. Racemic threo-3-methylaspartate was used in these experiments as the D-isomer has previously been shown to be neither a substrate nor an inhibitor of the enzyme [4].…”
Section: Methodsmentioning
confidence: 99%