Methionine synthase mechanism

Figure 12 Postulated reaction mechanism of methionine synthase (MetH) showing the interconversion of the various oxidation states of enzyme-cofactor species, E B12.

The cobalt center in MeCbl, one of the two important B12 coenzymes, is clearly involved in key steps in catalytic methyl transfer processes. Here, the Co center cycles between Co(I) and Co(III)CH3. In methionine synthase, the proposed mechanism involves direct nucleophilic attack on the C of the Co(III)CH3 group. In model reactions, the thiolate most frequently simply binds tram to the alkyl group to give a product recently established by an x-ray study of a model system. The protein may block access to the Co, thus preventing this reaction common in models. It is likely that the reactive form of the bound cofactor is five-coordinate in the key point in the catalytic cycle. This reactive form will lead to a four-coordinate Co(I) species. The axial coordination of the cofactor by a protein imidazole allows for a finer tuning of the Cbl chemistry and may permit control of the coordination number. Thus, recoordination of Co in the Co(I) state may facilitate attack on methyltetrahydrofolate and re-formation of Co(III)CH3. 

Eseapement meehanisms are real possibilities for any protein mechanism that uses oxidant indueed reduetion or reduetant induced oxidation to control the direetion of multiple eleetron transfer at cluster sites. Thus escapements may be active in mitoehondrial Complex 1 (Dutton et al., 1999 Dutton et al., 1998). They should also be eonsidered for anaerobic ribonucleotide reductase (see chapter by M. Sahlin and B-M. Sj berg) and in methionine synthase (see chapter by N. Marsh). 

The half-wave potential for the enzyme-bound Co VCo cobalamin couple of the methionine synthase from E. coli at 526 mV versus SHE is about 80 mV lower than that of the Co /Cokcobalamin couple in neutral aqueous solution. Access to the catalytic cycle of the enzyme by one-electron reduction of Co kcobalamin (and reactivation upon occasional adventitious formation of Co -cobalamin) is indicated to be accomplished by a unique mechanism. The (thermodynamically unfavorable) reduction with intermediate formation of the enzyme-bound Cokcobalamin is driven by a rapid methylation of the highly reduced Co -center of the reduced corrin with Y-adenosyhnethionine. The modular nature of methionine synthase allows for the control of the methyl-group transfer processes by modulating and alternating conformational equilibria. ... 

Cobalamin-dependent methionine synthase contains a built-in repair mechanism. If accidental oxidation of cob(I)alamin leads to inactive cob(lI)alamin, then the enzyme employs SAM and reduced flavodoxin to regenerate cob(I)alamin. Although the redox equilibrium below lies mainly on the left side, any cob(l)alamin formed is trapped by SAM-dependent methylation to yield methylcobalamin. 

In those cases where the methyl group donor is chemically less reactive, as with iV -methyl-THF and methanol, retention of configuration is observed (Table XII). For the cobalamin-dependent methionine synthase, retention of configuration is consistent with a postulated mechanism for this enzyme involving two sequential transfers of the methyl group, one from fV -methyI-THF to cobalt to generate methylcobalamin and a second from cobalt to the sulfur of homocysteine (349) [Eq. (66)] ... 

The methionine synthases represent a paradigm in organocobalamin biochemistry. The cobalamin-dependent protein. Met H, catalyzes the transfer of a methyl group from methyl-H4folate to Hcys via a MeCbl intermediate. Biophysical evidence is in accord with a mechanism involving... 

Wolthers KR, Lou X, Toogood HS, Leys D, Scrutton NS (2007) Mechanism of coenzyme binding to human methionine synthase reductase revealed through the crystal structure of the FNR-like module and isothermal titration calorimetry. Biochemistry 46 11833-11844... 

Conversion of the C-terminal fragment to an activation-competent conformation is the first of several rearrangements of methionine synthase that we would like to study. Computations would complement the structure determinations, in the best Lipscomb tradition, by examining not only the static pictures of various conformers but also the likely pathways for interconversion of the structures (52) and the mechanisms for activation of bound substrates (55). 

The overall picture which emerges for the mechanism of methionine synthase is summarized in Scheme 8.6, where HSR is homocysteine and only the reacting part of Me-H folate is shown. 

Homocystinuria can be treated in some cases by the administration of pyridoxine (vitamin Bs), which is a cofactor for the cystathionine synthase reaction. Some patients respond to the administration of pharmacological doses of pyridoxine (25-100 mg daily) with a reduction of plasma homocysteine and methionine. Pyridoxine responsiveness appears to be hereditary, with sibs tending to show a concordant pattern and a milder clinical syndrome. Pyridoxine sensitivity can be documented by enzyme assay in skin fibroblasts. The precise biochemical mechanism of the pyridoxine effect is not well understood but it may not reflect a mutation resulting in diminished affinity of the enzyme for cofactor, because even high concentrations of pyridoxal phosphate do not restore mutant enzyme activity to a control level. 

The metabolic control of methionine metabolism is complex and involves, for example, changes of enzyme levels in particular tissues, mechanisms linked to the kinetic properties of the various enzymes and their interaction with metabolic effectors [6, 7]. A particularly important metabolic effector is AdoMet. This inhibits the low Km isoenzymes of MAT, and MTHF reductase, inactivates betaine methyltransferase, but activates MAT III (the high-Km isoenzyme) and cystathionine /1-synthase. Therefore, high methionine intake and thus higher AdoMet levels favour trans-sulphuration, and when levels are low methionine is conserved. AdoHcy potently inhibits AdoMet-dependent methyltransferases and both Hey remethylating enzymes. Another important control mechanism is the export of Hey from cells into the extracellular space and plasma, which occurs as soon as intracellular levels increase [8]. 

Fig. 1. Ethylene biosynthesis. The numbered enzymes are (1) methionine adenosyltransferase, (2) ACC (l-aminocyclopropane-l-carboxylic acid) synthase, (3) ethylene forming enzyme (EFE), (4) 5 -methylthio-adenosine nucleosidase, (5) 5 -methylthioribose kinase. Regulation of the synthesis of ACC synthase and EFE are important steps in the control of ethylene production. ACC synthase requires pyridoxal phosphate and is inhibited by aminoethoxy vinyl glycine EFE requires 02 and is inhibited under anaerobic conditions. Synthesis of both ACC synthase and EFE is stimulated during ripening, senescence, abscission, following mechanical wounding, and treatment with auxins.

E-6) Homocysteinurla (defect in cystathionine synthase at this step). This is the most common form of ho-mocysteinuria). The enzyme defect leads to elevated levels of homocysteine, which can be detected in the urine. Serum methionine is also elevated. The clinical problems include dislocation of the lens, mental retardation, and various skeletal and neurologic problems. The mechanisms are unclear. Treatment may include administration of pyridoxine, decreasing dietary methionine and increasing cysteine. Elevated blood homocysteine is a risk factor for heart disease. 

Figure 3. The mechanism ofa irin s effect on platelets The 599 amino acid polypeptide chain of PGH synthase (center wavy line NHj-terminal methionine-1, COOH-terminal leucine-599) exerts cyclo-oxygenase activity as shown above (oxygenation/cycli-zation of arachidonate to PGGj) and interacts with aspirin as shown below. The serine residue located at position 529 of the polypeptide chain of cyclo-oxygenase is acetylated through transfer of aspirin s acetyl groiq> as indicated in bold face. Covalently-modified, acetylated PGH synthase carries a single acetyl group in its active site and lacks all cyclo-oxygenase activity.

Several PLP-dependent enzymes catalyze elimination and replacement reactions at the y-carbon of substrates, an unusual process which provides novel routes for mechanism-based inactivation. An example of this class of enzymes is cystathionine y-synthase [0-succinylhomoserine (thiol)-lyase], which converts (7-succinyl-L-homoserine and L-cysteine to cystathionine and succinate as part of the bacterial methionine biosynthetic pathway (Walsh, 1979, p. 823). Formation of a PLP-stabilized o-carbanion intermediate activates the )8-hydrogen for abstraction, yielding j8-carbanion equivalents and allowing elimination of the y-substituent. The resulting j8,y-unsaturated intermediate serves as an electrophilic acceptor for the replacement nucleophile. Suitable manipulation of the j8-carbanion intermediate allows strategies for the design of inactivators which do not affect enzymes which abstract only the a-hydrogen. 

Fig. II. Mechanism proposed for inactivation of cystathionine y-synthase and methionine y-lyase by 2-amino-4-chloro-5-(p-nitrophenylsulfinyl)pentanoic acid (11).

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