Methylmalonyl coenzyme A mutase

There is one exception to the rule that requires bulky hydrophobic residues to fill the interior of eight-stranded a/p barrels in order to form a tightly packed hydrophobic core. The coenzyme Biz-dependent enzyme methylmalonyl-coenzyme A mutase, the x-ray structure of which was determined by Phil Evans and colleagues at the MRC Laboratory of Molecular... 

Figure 4.4 Schematic diagram of the structure of the a/p-barrel domain of the enzyme methylmalonyl-coenzyme A mutase. Alpha helices are red, and p strands are blue. The inside of the barrel is lined by small hydrophilic side chains (serine and threonine) from the p strands, which creates a hole in the middle where one of the substrate molecules, coenzyme A (green), binds along the axis of the barrel from one end to the other. (Adapted from a computer-generated diagram provided by P. Evans.)...

Dong, S. L., Padmakumar, R., Maiti, N., Baneqee, R., and Spiro, T. G., 1998, Resonance raman spectra show that coenzyme B12 binding to methylmalonyl-coenzyme A mutase changes the corrin ring conformation but leaves the C06C bond essentially unaffected. J. Am. Chem. Soc. 120 994799948. 

The answer is d. (Murray, pp 238-249. Scriver, pp 2165-2194. Sack, pp 121-144. Wilson, pp 287-324.) Propionic acidemia (232000) results from a block in propionyl CoA carboxylase (PCC), which converts propionic to methylmalonic acid. Excess propionic acid in the blood produces metabolic acidosis with a decreased bicarbonate and increased anion gap (the serum cations sodium plus potassium minus the serum anions chloride plus bicarbonate). The usual values of sodium (-HO meq/L) plus potassium ( 4 meq/T) minus those for chloride (-105 meq/L) plus bicarbonate (—20 meq/L) thus yield a normal anion gap of -20 meq/L. A low bicarbonate of 6 to 8 meq/L yields an elevated gap of 32 to 34 meq/L, a gap of negative charge that is supplied by the hidden anion (propionate in propionic acidemia). Biotin is a cofactor for PCC and its deficiency causes some types of propionic acidemia. Vitamin B deficiency can cause methylmalonic aciduria because vitamin Bn is a cofactor for methylmalonyl coenzyme A mutase. Glycine is secondarily elevated in propionic acidemia, but no defect of glycine catabolism is present. 

Methylmalonic acid (MMA) is a metabolic intermediate in the biosynthesis of succinic acid from propionic acid, a step that involves the enzyme Methylmalonyl Coenzyme A mutase and a vitamin B12-derived cofactor. MMA concentrations increase when vitamin B12 is deficient hence, MMA can be used as a clinical biomarker of vitamin B12 status. In addition, mutations in the genes encoding the enzyme responsible for MMA metabolism or the enzymes responsible for vitamin B12 metabolism can lead to heritable disorders, known as methylmalonic acidemias. 

The substrate analogue ethylmalonyl coenzyme A has been used to obtain further information concerning the mechanism of the methylmalonyl-coenzyme A mutase reaction. ... 

Coenzyme Bj2 (shortened ACH2-Bj2) is a cofactor in various enzymatic reactions. Reaction (7.2.10) is an example. The enzyme is methylmalonyl-coenzyme A mutase the migrating group is framed. For additional and more complete information, see the article by J. Halpem. ... 

Adenosylcobalamin (coenzyme B 2) is required in a number of rearrangement reactions that occurring in humans is the methylmalonyl-Co A mutase-mediated conversion of (R)-methylmalonyl-Co A (6) to succinjl-CoA (7) (eq. 1). The mechanism of this reaction is poorly understood, although probably free radical in nature (29). The reaction is involved in the cataboHsm of valine and isoleucine. In bacterial systems, adenosylcobalamin drives many 1,2-migrations of the type exemplified by equation 1 (30). 

Vitamin B12 is a biologically active corrinoid, a group of cobalt-containing compounds with macrocyclic pyrrol rings. Vitamin B12 functions as a cofactor for two enzymes, methionine synthase and L-methylmalonyl coenzyme A (CoA) mutase. Methionine synthase requires methylcobalamin for the methyl transfer from methyltetrahydrofolate to homocysteine to form methionine tetrahy-drofolate. L-methylmalonyl-CoA mutase requires adenosylcobalamin to convert L-methylmalonyl-CoA to succinyl-CoA in an isomerization reaction. An inadequate supply of vitamin B12 results in neuropathy, megaloblastic anemia, and gastrointestinal symptoms (Baik and Russell, 1999). 

The catalytic action of vitamin B12 coenzyme in the enzymatic conversion of methylmalonyl-coenzyme A to succinyl-coeiizyme A and of related mutases is as yet unexplained and poses fascinating problems (Equations 2- 5). 

Vrilbloed JW, Zerbe-Brnkhardt K, Ratnatilleke A, Grubelnik-Leiser A, Robinson JA (1999) Insertional inactivation of methylmalonyl coenzyme A (CoA) mutase and isobutyryl-CoA mutase genes in Streptomyces cinnamonensis-. influence on polyketide antibiotic biosynthesis. J Bacteriol 181 5600 - 5605... 

Mancia, E., et al. How coenzyme Biz radicals are generated the crystal structure of methylmalonyl-coen-zyme A mutase at 2 A resolution. Strueture 4 339-350, 1996. 

Methylmalonyl-CoA mutase 5 -deoxyadenosylco-balamin is part of dimethylbenzimidazolecobamide coenzyme, a constituent of methylmalonyl-CoA mutase. This mutase catalyses the isomerization of methylmalonyl-CoA to succinyl-CoA (anaplerotic reaction of the citric acid cycle). 

R) -2-Methyl-3-oxopropanoyl-coenzyme A, METHYLMALONYL-CoA EPIMERASE l-METHYLMALONYL-CoA MUTASE... 

Figure 16-23 Three-dimensional structure of methylmalonyl-CoA mutase from Propionobacterium. shermanii. The B12 coenzyme is deeply buried, as is the active site. A molecule of bound desulfo-coenzyme A, a substrate analog, blocks the active site entrance on the left side. From Mancia et al.i05 Courtesy of Philip R. Evans.

As a result of the reduced activity of the mutase in vitamin B12 deficiency, there is an accumulation of methyhnalonyl CoA, some of which is hydrolyzed to yield methylmalonic acid, which is excreted in the urine. As discussed in Section 10.10.3, this can be exploited as a means of assessing vitamin B12 nutritional status. There may also be some general metabolic acidosis, which has been attributed to depletion of CoA because of the accumulation of methyl-malonyl CoA. However, vitamin B12 deficiency seems to result in increased synthesis of CoA to maintain normal pools of metabolically useable coenzyme. Unlike coenzyme A and acetyl CoA, neither methylmalonyl CoA nor propionyl CoA (which also accumulates in vitamin B12 deficiency) inhibits pantothenate kinase (Section 12.2.1). Thus, as CoA is sequestered in these metabolic intermediates, there is relief of feedback inhibition of its de novo synthesis. At the same time, CoA may be spared by the formation of short-chain fatty acyl carnitine derivatives (Section 14.1.1), which are excreted in increased amounts in vitamin B12 deficiency. In vitamin Bi2-deficient rats, the urinary excretion of acyl carnitine increases from 10 to 11 nmol per day to 120nmolper day (Brass etal., 1990). 

ATP to yield the d isomer of methylmalonyl CoA (Figure 22.11). This carboxylation reaction is catalyzed by propionyl CoA carboxylase, a biotin enzyme that is homologous to and has a catalytic mechanism like that of pyruvate carboxylase (Section 16.3.2). The d isomer of methylmalonyl CoA is racemized to the 1 isomer, the substrate for a mutase that converts it into succinyl CoA by an intramolecular rearrangement. The -CO-S-CoA group migrates from C-2 to C-3 in exchange for a hydrogen atom. This very unusual isomerization is catalyzed by methylmalonyl CoA mutase, which contains a derivative of vitamin Bj2, cobalamin, as its coenzyme. 

The transamination of P-aminoisobutyrate to form methylmalonate semialdehyde requires pyridoxal phosphate as a cofactor. This reaction is similar to the conversion of ornithine to glutamate y-semialdehyde. Then NAD+ serves as an electron acceptor for the oxidation of methylmalonate semialdehyde to methylmalonate. The conversion of methylmalonate to methylmalonyl CoA requires coenzyme A. The final reaction, in which methylmalonyl CoA is converted to succinyl CoA, is catalyzed by methylmalonyl CoA mutase, an enzyme that contains a derivative of vitamin B12 as its coenzyme. 

Finally, it should be noted that coenzyme B g would probably have been discovered by Beck et during their studies of the conversion of methylmalonyl-CoA to succinyl-CoA by the sheep kidney mutase (isomerase) were it not for the fact that this enzyme binds the coenzyme very tightly in such a way that it is not susceptible to inactivation by light. Because of these properties they were unable to obtain a coenzyme-free mutase and so concluded that the reaction probably has no cofactor requirement. 

It appears that the methane synthesis and transmethylation functions of Methanobacillus kuzneccovii can be separated by removal of particulate matter from cell-free extracts, a procedure which leaves only the transferase activity. The methane synthesizing ability is not, however, destroyed and is reinstated on mixing the separated fractions. Measurement of enzyme acti ty of the methyl-malonyl coen me A mutase system with and without added coenzyme Bia in the leucocytes of patients with methylmalonyl acidaemia is of value in diagnosing two variants of the disease. ... 

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