| Cobalamin-dependent methionine synthase catalyzes methyl group transfer from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine, with the cobalamin prosthetic group serving as an intermediate methyl carrier. The enzyme is susceptible to inactivation by the anaesthetic gas nitrous oxide, through either short-term exposure to high levels or chronic exposure to low levels of this agent. The goal of this work was to develop a biochemical model to explain how the enzyme becomes inactivated under an atmosphere of nitrous oxide. The chemical reaction between the reactive, enzyme-bound cob(I)alamin intermediate and nitrous oxide was investigated, and specific sites on the enzyme that become modified during the inactivation were identified. This was achieved by reducing the cobalamin prosthetic group to cob(I)alamin in an electrochemical cell under an atmosphere of nitrous oxide. Degradation of nitrous oxide in the cell apparently releases a damaging oxidant, proposed to be hydroxyl radical, and the observed modifications provide insight into the spatial relationship between the reactive cobalamin prosthetic group and regions of the enzyme that it must contact. Because methionine synthase is an exceptionally large monomer of 136.1 kDa, we used proteolysis to separate the enzyme into domains that were characterized using a combination of electrospray mass spectrometry and N-terminal sequence analysis. A C-terminal, 37.7 kDa domain was isolated that bound the S-adenosylmethionine coenzyme required for enzyme activation, and this proved to be the primary site of oxidative damage. The complementary, N-terminal domain of 98.4 kDa retained the bound cobalamin, and this domain was able to catalyze methyl transfer from 5-methyltetrahydrofolate to homocysteine. We have proposed and tested a model to explain how the domains of methionine synthase function together to effect catalysis, and this model provides an explanation for why the C-terminal domain that becomes modified must approach the cobalamin, the site where nitrous oxide is reductively degraded. |
