©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Demonstration That Mammalian Methionine Synthases Are Predominantly Cobalamin-loaded (*)

(Received for publication, May 4, 1995; and in revised form, June 8, 1995)

Zhiqiang Chen Sarbani Chakraborty Ruma Banerjee (§)

From the Biochemistry Department, University of Nebraska, Lincoln, Nebraska 68588-0664

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)and, in so doing, recycles methionine. It also functions to free up the circulating form of folic acid, CH(3)-H(4)-folate, which is delivered from the blood stream to the cells, to a more generally usable form, H(4)-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(3)-H(4)-folate (present predominantly as the monoglutamate(3) ) is poor and because CH(3)-H(4)-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(3)-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(3)-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.


EXPERIMENTAL PROCEDURES

Materials

The following materials were purchased from Sigma: AdoMet, OH-cobalamin, DTT, FMN, and FAD. Titanium chloride was purchased from Aldrich, and titanium citrate was prepared as described previously(14) . (6R,6S)-5-[^14C]CH(3)-H(4)-folate (barium salt, 55 mCi/mmol) was purchased from Amersham Corp. (6R,6S)-CH(3)-H(4)-folate (calcium salt) was obtained from Schircks' Laboratories (Switzerland). The polyclonal antibodies to porcine methionine synthase were generated in rabbits at the Cleveland Clinic Foundation in the laboratory of Dr. Donald Jacobsen.

Purification of Methionine Synthase

Porcine methionine synthase was purified as described previously(12) , except that OH-cobalamin was not added to the enzyme or buffers at any stage of the purification. Human methionine synthase was purified from a placenta obtained within minutes of delivery, cubed, and stored frozen at -80 °C until the time of purification. The human enzyme was purified through the first four steps of the protocol described previously(12) . Protein concentrations were determined by the Bio-Rad protein assay based on the method of Bradford (15) with bovine serum albumin as the standard.

Anaerobic Methionine Synthase Assay

The assays were performed in 10-ml serum vials in which the atmosphere had been exchanged for a N(2)/H(2) (19:1) mixture in a Coy anaerobic chamber. All stock reagent solutions were made anaerobic by bubbling nitrogen through them for at least 30 min and were stored in stoppered serum vials. This procedure was repeated periodically with solutions that were stored for long periods of time. The enzyme (placed in a stoppered serum vial on ice) was made anaerobic by gently passing nitrogen over the solution for 30 min. The reaction mixture contained the following: 100 mM potassium phosphate buffer, pH 7.2, 500 µM homocysteine, 19 µM AdoMet, 2 mM titanium citrate or 25 mM DTT, 250 µM (6R,6S)-5-[^14CH(3)]-H(4)-folate (2000 dpm/nmol), and enzyme in a final volume of 1 ml. The reaction was run either in the presence or absence of OH-cobalamin (50 µM). The mixture lacking CH(3)-H(4)-folate was preincubated at 37 °C for 5 min. The reaction was initiated with CH(3)-H(4)-folate, incubated for 10 min at 37 °C, and terminated by heating at 98 °C for 2 min. Beyond this point, the reaction vials were uncapped, and the solutions were handled aerobically. The assay mixture was cooled on ice and then passed through a 0.5 6 cm column of Dowex 1 X8 (chloride form). The column was washed with 2 ml of water, and the eluate from the sample loading and rinse was collected in a scintillation vial. A biodegradable scintillation fluid, Ecolite, (10 ml) was added to the aqueous sample and counted. All reported values are corrected for the counts observed in control assays run in parallel from which enzyme had been omitted.

Anaerobic Assay of Methionine Synthase Using FMNH(2)or FADHas a Reductant

Solutions of FMN and FAD (2 mM each) in 5 ml of water containing platinum oxide (30 mg) were reduced by bubbling with hydrogen gas for 1.5 h. Complete reduction was indicated by the disappearance of the 450-nm UV-visible absorption band of the oxidized flavins. The assays were then conducted as described above with the exception that reduced flavin (0.1 mM) replaced DTT or titanium citrate and a stream of hydrogen was passed over the assay mixtures during the course of the reaction.

Estimation of the Ambient Potential of the in Vitro Assays

The redox potentials of the DTT/OH-cobalamin and titanium citrate assays were estimated spectrophotometrically. Anaerobic solutions of the reagents were mixed in a sealed cuvette in which the atmosphere had been replaced with nitrogen. To estimate the potential of the DTT/OH-cobalamin assay, the following components were mixed: DTT (25 mM), OH-cobalamin (50 µM), benzylviologen (100 µM) in 1 ml of anaerobic 100 mM phosphate buffer, pH 7.2. The samples were scanned between 350 and 700 nm. The concentration of the reduced benzylviolgen was estimated from the absorption at 555 nm, using an extinction coefficient of 10 mM cm. To estimate the potential of the titanium citrate assay, titanium citrate (2 mM) and methyl viologen (100 µM) were mixed in 1 ml of anaerobic 100 mM phosphate buffer, pH 7.2. The concentration of the reduced methyl viologen was estimated from the absorption at 604 nm, using an extinction coefficient of 13.6 mM cm(16) .


RESULTS AND DISCUSSION

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, (^2)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. (^3)Thus, the amount of apoenzyme is overestimated. We have found that both FADH(2) and FMNH(2) 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. (^4)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) . (^5)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.


FOOTNOTES

*
This work was supported in part by Grant DK45776 from the National Institutes of Health (to R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 402-472-2941; Fax: 402-472-7842; RBANERJEE{at}crcvms.unl.edu.

(^1)
The abbreviations used are: AdoMet, S-adenosylmethionine; DTT, dithiothreitiol.

(^2)
The midpoint potential of the titanium(III) citrate/titanium(IV) citrate couple is pH-dependent and has been reported to be -480 mV at pH 7 (4).

(^3)
This assay employs thiols, and during long incubations it is possible that some of the propylcobalamin decomposes to OH-cobalamin, which would then mimic the standard assay.

(^4)
In the methods described in the literature, the flavins are reduced by bubbling with H(2) for only 10 min. In our experience, this leads to incomplete reduction as judged by UV-visible absorption spectroscopy. The assay with reduced flavins have been used most recently by Kolhouse and co-workers (5, 6), but precautions were apparently not taken to maintain good anaerobiosis in the reaction mixture. Based on our results, this would lead to an overestimation of the apoenzyme form accounting for their low estimate of the holoenzyme (10-40%).

(^5)
Comparison of the specific activities of the partially purified (0.53 µmol of methionine/min/mg of protein) and completely purified (1.7 µmol of methionine/min/mg of protein) enzymes indicated that the enzyme used for spectroscopic analysis was 31% pure. The concentration of porcine methionine synthase in the sample was then calculated using a molecular mass of 155 kDa (12).


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.