Nitrous oxide inactivation of cobalamin-dependent methionine synthase from Escherichia coli: characterization of the damage to the enzyme and prosthetic group.
Nitrous oxide, or laughing gas, is an anaesthetic agent that inactivates cobalamin-dependent methionine synthase. This enzyme uses the highly reactive, enzyme-bound cob(I)alamin oxidation state of the prosthetic group to effect methyl group transfer from 5-methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. The cob(I)alamin is capable of reductively degrading nitrous oxide, and here we characterize the modifications that occur to the Escherichia coli enzyme following electrochemical inactivation. Methionine synthase was inactivated on a milligram scale by equilibrating enzyme containing bound cob(II)alamin with a reduced electrochemical mediator to give the reactive cob(I)alamin state under an anaerobic atmosphere of nitrous oxide. The primary damage occurs to a 37.2-kDa domain that binds S-adenosylmethionine (AdoMet), and inactive enzyme can no longer be reductively methylated using AdoMet. The damage is oxidative, and it includes the covalent addition of the mediator, triquat, to the enzyme selectively at valine 1177, as well as the formation of a covalent cross-link between peptides containing the only two cysteines within this domain. Spectrally, the prosthetic group bound to inactive enzyme resembles cob(II)alamin, although some loss in absorbance is apparent. When the enzyme was reconstituted with [57Co]cobalamin and the inactivation repeated, the cobalamin was recovered unmodified in approximately 75% yield, but two products derived from the cobalamin were also observed. We interpret the finding of oxidatively modified products as strong evidence that reductive degradation of nitrous oxide releases a potent oxidant, presumably hydroxyl radical or its equivalent, that is capable of modifying sites proximal to the cobalamin.
Drummond
J T
JT
Biophysics Research Division, University of Michigan, Ann Arbor 48109.
Matthews
R G
RG
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000187:drug effects; D004563:Electrochemistry; D004926:Escherichia coli / Q000201:enzymology*; D006710:Homocysteine / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009609:Nitrous Oxide / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D012436:S-Adenosylmethionine / Q000378:metabolism; D013763:Tetrahydrofolates / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014805:Vitamin B 12 / Q000737:chemistry / Q000494:pharmacology*
D010446:Peptide Fragments; D013763:Tetrahydrofolates; D006710:Homocysteine; C030371:5,6,7,8-tetrahydrofolic acid; D012436:S-Adenosylmethionine; D008715:Methionine; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D014357:Trypsin; D009609:Nitrous Oxide; D014805:Vitamin B 12; C005984:5-methyltetrahydrofolate