(Received for publication, May 4, 1995; and in revised form, June 8, 1995)
From the
Methionine synthase is an important cellular housekeeping enzyme
and is dependent on the cofactor cobalamin, a derivative of vitamin
B, for activity. It functions in two major metabolic
pathways including the tetrahydrofolate-dependent one-carbon cycle and
the salvage pathway for methionine. Its dysfunction has several
physiological ramifications and leads to the development of
megaloblastic anemia. In addition, it is suspected to be involved in
the pathogenesis of neural tube defects. An issue that is central in
weighing therapeutic options for methionine synthase-related disorders
is the extent to which the enzyme exists as apoenzyme in vivo and, thus, can be potentially responsive to vitamin B
therapy. Despite the importance of this issue, the extent of
holo- versus apoenzyme in mammalian tissue is controversial
and unresolved. To address this question, we have developed a
convenient anaerobic assay that employs titanium citrate to deliver low
potential electron equivalents. The reductive activation of this enzyme
is essential under in vitro assay conditions. We find that
both the human placental and porcine liver methionine synthases exist
predominantly in the holoenzyme form (90-100%) in the crude
homogenate. In addition, the activity of the pure enzyme measured in
the titanium citrate assay is also independent of exogenous cofactor,
revealing that the cobalamin is tightly bound to the active site.
Methionine synthase (EC 2.1.1.13) is a cytoplasmic enzyme that
straddles two major metabolic pathways. It remethylates homocysteine, a
toxic catabolite of S-adenosylmethionine (AdoMet) ()and, in so doing, recycles methionine. It also functions
to free up the circulating form of folic acid,
CH
-H
-folate, which is delivered from the blood
stream to the cells, to a more generally usable form,
H
-folate. Its activity is dependent on the cofactor,
cobalamin, a derivative of vitamin B
.
Metabolic
repercussions of methionine synthase dysfunction include depletion of
AdoMet and methionine pools with concomitant elevation of homocysteine,
a suspected cardiovascular risk factor ( (1) and (2) and references therein). It also lowers the folate pool
because intracellular retention of CH-H
-folate
(present predominantly as the monoglutamate(3) ) is poor and
because CH
-H
-folate is not a substrate for any
other known enzyme. Thus, pleiotropic effects on both the sulfur- and
one carbon-dependent cell cycles ensue. The clinical condition is
characterized by megaloblastic anemia(4) .
An important unresolved and controversial issue concerning methionine synthase is how much of the enzyme in the cell is present as holoenzyme, i.e. has the cofactor bound to it. Resolution of the relative levels of holo- versus apoenzyme in the cell is complicated by the mixed oxidation states of cobalamin in the isolated methionine synthase and the need for a relatively low potential single electron donor to activate the protein. Thus, the estimates of the proportion of holoenzyme have been conflicting, ranging from 10% (5, 6) to 100%(7) .
Methionine synthase can
be potentially isolated in four stable forms depicted as species I-IV in Fig. S1. Of these, three are
variations of holoenzyme, in which the bound cobalamin is either in an
``active form'' (IV, CH-cobalamin) or in
an ``inactive form'' (II and III). The latter
two species can be readmitted to the catalytic cycle in a reaction that
is independent of added cobalamin but dependent on a reducing system
and AdoMet. Recruitment of the apoenzyme form (I) to the
catalytic cycle is dependent, in addition, on cobalamin.
Figure S1: Scheme 1Depiction of the various oxidation states of methionine synthase and the requirements of each form for their participation in the catalytic cycle. DMB refers to the intramolecular base, dimethylbenzimidazole.
Several different assays have been employed to monitor methionine synthase activity and to estimate the proportion of holo- versus apoenzyme(5, 6, 8, 9, 10, 11, 12) . These include the use of either reduced flavins(5, 6, 8, 10) , thiols and OH-cobalamin (the standard assay) (12, 13) or thiols and the inhibitor, propylcobalamin(9, 11) . Of these, only the assay utilizing reduced flavin as an electron source can provide an appropriate measure of the holo- and apoenzyme forms. It is, however, tedious and depends on the need for freshly prepared reduced flavins along with spectral monitoring to ensure complete reduction of the flavin. In the absence of these precautions, variable concentrations of reduced flavin are present in the assay mixture leading to an overestimation of the apoenzyme concentration.
The
problem with the standard assay that is routinely employed to monitor
methionine synthase activity is that it relies on the supply of
exogenous cobalamin (usually in the form of OH-cob(III)alamin) and a
reductant such as dithiothreitol. Hydroxocobalamin potentially plays a
dual role. It is required in delivering one-electron equivalent to
forms II and III, and it can bind to the apoenzyme form (I), converting it to holoenzyme. Although the specific route of
electron transfer is not understood, omission of OH-cobalamin from the
assay short circuits the delivery of electron equivalents to the
enzyme. This can lead to a gross underestimation of enzyme
activity(12) . This is because (i) the fraction of holoenzyme
containing OH-cobalamin (II) or cob(II)alamin (III) is
excluded from the catalytic cycle, and (ii) active enzyme initially
present as CH-cob(III)alamin (IV) accumulates in an
inactive form (III) due to oxidative escape of cob(I)alamin (I) from the turnover cycle without a functional avenue for
reactivation. Hence, an accurate estimation of the proportion of holo- versus apoenzyme must rely on a system where single electron
donation is afforded via a route other than exogenous
cobalamin. In this study, we describe the development of an anaerobic
assay that meets this criterion. This has led us to the surprising
finding that the mammalian methionine synthases from both porcine liver
and human placenta exist predominantly in the holoenzyme form.
Moreover, the cofactor is tightly bound and is retained during multiple
purification steps.
We have developed an anaerobic assay for methionine synthase that permits evaluation of the extent of apo- versus holoenzyme without some of the ambiguities of the previously published procedures. The assay provides reducing equivalents from titanium citrate to activate methionine synthase, thus bypassing the need for exogenous cobalamin in this role. Under these conditions, only the holoenzyme forms of methionine synthase (, II-IV) contribute to catalytic turnover. In the presence of titanium citrate and exogenous OH-cobalamin, both apoenzyme and holoenzyme forms (I-IV) participate in catalytic turnover. Thus, comparison of activities in the presence and absence of OH-cobalamin provides an estimate of the percentage of apoenzyme (I).
To establish that titanium citrate does not adversely
affect methionine synthase, the activity of the enzyme was compared in
the presence of DTT/OH-cobalamin or titanium citrate, both under
anaerobic conditions. As can be seen from Table 1, titanium
citrate does not inhibit methionine synthase. In fact, enzyme activity
estimated in the presence of titanium citrate is 1.5-fold higher
than that in the standard assay. This is presumably due to the lower
reducing potential of titanium citrate versus the
DTT/OH-cobalamin system. Our estimates of the ambient redox potential
of the DTT/OH-cobalamin and titanium citrate assay mixtures are
approximately -350 and -450 mV, (
)respectively, versus the standard hydrogen electrode. The reductive
activation cycle renders the activity of methionine synthase responsive
to the ambient redox potential. The mechanism of reductive activation
is postulated to be a one-electron reduction of cob(II)alamin to
cob(I)alamin that is rapidly trapped by an exergonic methylation
reaction with AdoMet(17, 18, 19) .
Alternative mechanisms involving a sulfur radical cation intermediate
on AdoMet have also been suggested(13) . Although the midpoint
potential of cobalamin bound to a mammalian methionine synthase is
unknown, it is likely to be comparable with that of the Escherichia
coli enzyme at -526 mV(19) . The lower ambient
potential provided by titanium citrate provides a greater driving force
for reduction. This would lead to more efficient return of oxidatively
ruined enzyme to the catalytic cycle and result in higher enzyme
activity.
The data presented in Table 1demonstrate that OH-cobalamin does not increase the activity of methionine synthase in either the cell extract or the purified state when the titanium citrate assay is employed. In contrast, when DTT is employed as a reductant under anaerobic conditions, a 4-fold diminution in the activity is observed in the absence of OH-cobalamin. These results indicate that the porcine methionine synthase exists predominantly in the holoenzyme form in the crude homogenate and that the enzyme retains its cofactor during the five purification steps. These results also demonstrate that the enzyme is isolated in several holoenzyme states. Apparently a fourth of the enzyme in both the crude and pure states has bound methylcobalamin and is thus active in catalysis even in the absence of a functional activation system. Under standard aerobic assay conditions, there is no detectable activity in the absence of OH-cobalamin (Table 2), presumably due to rapid and irreversible accumulation of enzyme in the inactive forms II and III. The data presented in Table 1and Table 2confirm the electron transfer role played by cobalamin in these assays. Thus, in the presence of thiols, exogenous cobalamins are required for reductive activation accounting for the observed 4-fold increase in activity. A similar role for cobalamin has been reported previously for the methylreductase system from Methanobacterium bryantii(20) . Oxidation of thiols by corrinoids have been demonstrated in nonbiological systems previously(21, 22) .
Next, enzyme activity from several different porcine livers was measured to determine if holoenzyme levels vary in individual animals (Table 2). In every instance, 90-100% of the enzyme activity is observed in the absence of exogenous cofactor, indicating that the enzyme is predominantly holoenzyme. The activities of porcine enzyme from five independent preparations are presented in Table 2.
We have
extended this study to the human enzyme from placenta, which was
previously purified and reported to be 10-40%
apoenzyme(5) . We purified the human enzyme 2000-fold and
have found that, like the porcine enzyme, it is present predominantly
in the holoenzyme form (Table 2). In addition, Western analysis
of the partially purified human enzyme with antibodies generated
against the porcine protein, reveals a single cross-reacting band of
identical molecular mass (
155 kDa) by denaturing polyacrylamide
gel electrophoresis (Fig. 1). This is in contrast to an earlier
report (5) that the human placental enzyme is a heterotrimer
with subunit molecular masses of 90, 45, and 35 kDa, respectively. The
restricted availability of human tissue to us has limited the sample
size in this study to a single placenta. However, the results from both
Western analysis as well as enzyme activity assays support our
conclusion that the human methionine synthase is comparable to the more
extensively characterized porcine enzyme. Our results are consistent
with an earlier study by Taylor and co-workers (7) on
methionine synthase activity in normal and megaloblastic bone marrows.
In normal marrow cells, enzyme activity was not stimulated in the
presence of exogenous cobalamin, indicating that 100% of the enzyme was
present as holoenzyme. This was in contrast to marrow from patients
with pernicious anemia characterized by very low serum B
,
where only 13% of the methionine synthase was holoenzyme.
Figure 1:
SDS-PAGE and Western analysis of
porcine and human methionine synthases. A, SDS-PAGE of
methionine synthase from pig liver (lane1, 10
µg) and human placenta (lane2, 20 µg). The
gel was stained with Coomassie Blue. In both protein samples, the
methionine synthase has been purified approximately 1500-fold. The leftlane has protein standards from Bio-Rad and
their molecular masses in kDa are indicated. B, detection of
porcine (1) and human (2) methionine synthases by
immunological staining with antibodies raised against the porcine
methionine synthase. A second gel was run in parallel with that shown
in A and transferred to a nitrocellulose membrane. Although
approximately equal amounts of the porcine and human methionine
synthases were loaded, the antibody cross-reacts with the porcine
antigen more intensely.
There are
at least two methods that have been widely employed to determine the
relative proportions of holo- versus apomethionine synthase
concentration. The first uses the inhibitor propylcobalamin, in the
presence of thiols(9, 11) . The second uses reduced
flavins as an electron source(6, 8, 10) . In
the first assay, there is no direct avenue for reductive activation of
the inactive holoenzyme. ()Thus, the amount of apoenzyme is
overestimated. We have found that both FADH
and FMNH
are comparable with titanium citrate in their ability to activate
methionine synthase (Table 3). In addition, the activity of the
enzyme with reduced flavins is not increased by adding OH-cobalamin,
indicating the predominance of holoenzyme. Although reduced flavins are
efficient in reductive activation, these assays are cumbersome and
susceptible to significant errors. The need to reduce the flavins by
bubbling hydrogen over 1-2 h as well as the maintenance of
anaerobiosis by passing a stream of hydrogen gas over the individual
assay mixtures is tedious and limits the number of assays that can be
readily performed. In addition, incomplete reduction of flavins prior
to their use in the assays leads to variability in the results and an
overestimation of the apoenzyme form. (
)It is thus critical
to establish spectrophotometrically that reduction of the flavins is
complete before they are employed in the assay. In contrast, titanium
citrate is easily prepared and, if handled properly, has a long shelf
life, and the assays are highly reproducible.
The kinetic studies
described above indicate that methionine synthase is predominantly in
the holoenzyme form. We have also examined the UV-visible spectrum of
the enzyme purified in the absence of exogenous cobalamin. The enzyme
was found to contain 1 mol of cobalamin/mol of enzyme using extinction
coefficients for OH-cobalamin of 20.2 mM cm
and 8.17 mM
cm
at 358 and 536 nm,
respectively(23) . (
)The spectrum is identical to
that reported previously for enzyme purified in the presence of
OH-cobalamin (added to the buffers(12) ). Thus, like the
bacterial enzyme, the mammalian methionine synthase also binds its
cofactor tightly(24) .
In conclusion, we have developed a
convenient anaerobic assay for methionine synthase and have found that
both the porcine liver and human placental enzymes are present
predominantly in the holoenzyme form and that the cofactor is tightly
bound. Methionine synthase plays a central role in cellular
housekeeping as it straddles two major metabolic cycles. While recent
clinical studies suggest a correlation between impaired methionine
synthase and neural tube
defects(25, 26, 27) , its role in
megaloblastic anemia and homocystinuria are well established. As
vitamin B therapies are considered in treatment of such
disorders, it will be germane to evaluate the extent to which the
enzyme is present as apoenzyme and could potentially thus be responsive
to higher doses of the cofactor.