Liver betaine-homocysteine S-methyltransferase activity undergoes a redox switch at the active site zinc

Abstract

Using a redox-inert methyl acceptor, we show that betaine-homocysteine S-methyltransferase (BHMT) requires a thiol reducing agent for activity. Short-term exposure of BHMT to reducing agent-free buffer inactivates the enzyme without causing any loss of its catalytic zinc. Activity can be completely restored by the re-addition of a thiol reducing agent. The catalytic zinc of BHMT is bound by three thiolates and one hydroxyl group. Thiol modification experiments indicate that a disulfide bond is formed between two of the three zinc-binding ligands when BHMT is inactive in a reducing agent-free buffer, and that this disulfide can be readily reduced with the concomitant restoration of activity by re-establishing reducing conditions. Long-term exposure of BHMT to reducing agent-free buffer results in the slow, irreversible loss of its catalytic Zn and a corresponding loss of activity. Experiments using the glutamate-cysteine ligase modifier subunit knockout mice Gclm(-/-), which are severely impaired in glutathione synthesis, show that BHMT activity is reduced about 75% in Gclm(-/-) compared to Gclm(+/+) mice.

Figure 1

A) Repletion of apoBHMT with exogenous zinc (Zn). Filled circles represent specific activity of apoenzyme (0.97 nmol) after repletion with increasing concentrations of exogenous Zn chloride. Error bars represent mean ± SD. Triplicates were tested for each point. B) Stimulated release of Zn from WT BHMT by 1mM methylmethane thiosulfonate (white circles) or 1mM hydrogen peroxide (white triangles) in reducing agent-free buffer and stimulated release of Zn from WT BHMT in buffer containing reducing agents (grey circles). Stimulated release of zinc from the enzyme was measured using the Zn chelate, 4-(2-pryidylazo)resorcinol (PAR), as described in Experimental Procedures.

Figure 2

Time-dependent loss of BHMT activity when stored in the absence of a thiol reducing agent. Enzyme (0.67 nmol) was assayed using the Bet-Hcy substrate pair 100% activity corresponds to a specific activity of 2050 nmol Met/hour/mg of BHMT. The MMTS-stimulated release of Zn from enzyme (3.5 nmol) was measured using PAR, and the Zn:BHMT monomer ratio is given in parenthesis for each selected time point. Exact details are given in Experimental Procedures.

Figure 3

Modifiable sulfhydryl groups on reduced and oxidized BHMT:5C→A enzyme. The number of free sulfhydryl groups per nmol of purified protein was measured using 5’,5’-dithio-bis-(2-nitro)benzoic acid (DTNB). The BHMT solution was made devoid of reducing agent by desalting chromatography and the number of modifiable –SH groups were measured right after βME depletion (white squares), one hour after βME depletion (gray dots) and after re-addition of βME followed by desalting chromatography (black triangles).

Figure 4

Relative ability of different thiol and non-thiol reducing agents to activate oxidized BHMT. Wild type enzyme (6.6 nmol) was assayed (6 h) using the DMSA-Asp substrate pair as described in the Experimental Procedures. Inset: Effect of Cys concentration on BHMT activity. Wild type enzyme (15 nmol) was assayed (6 h) using the DMSA-Asp substrate pair as described in the Experimental Procedures.