Methylmalonyl-CoA mutase activity

Vitamin deficiency can cause a megaloblastic anemia of the same type seen in folate deficiency (discussed in Chapter 17). In a patient with megaloblastic anemia, it is important to determine the underlying cause because Bjj defidency, if not corrected, produces a peripheral neuropathy owing to aberrant fatty acid incorporation into the myelin sheets associated with inadequate methylmalonyl CoA mutase activity. Excretion of methylmalonic acid indicates a vitamin Bjj deficiency rather than folate. 

E. Vitamin is a cofactor in two biochemical reactions, the conversion of homocysteine to methionine by the enzyme methionine synthase and the conversion of L-methylmalonyl-CoA to succinyl-CoA by methytmalonyl-CoA mutase. N -methyl THF is a methyl donor in the methionine synthase reaction. A folate deficiency would result in decreased methionine synthase activity and decreases in methionine and cystathionine concentrations, while homocysteine levels would be increased. A vitamin deficiency would also yield these same results, but in addition methytmalonate levels would increase as a consequence of a decrease in the activity of methylmalonyl-CoA mutase activity. 

In vitamin Bi2-deficient rats, the urinary excretion of methylmalonate is markedly increased, and the levels of methylmalonyl CoA mutase activity in liver and kidney homogenates, as well as the concentrations of deoxyadenosylcobalamin, are decreased in various tissue. The levels of deoxyadenosylcobalamin may be inadequate for full saturation of the methylmalonyl... 

Investigators studying the effect of vitamin deficiency in animal models are faced with the technical difficulty of producing the deficiency syndrome in experimental animals. Rats, pigs and other laboratory animals do not develop clean-cut megaloblastic anemia when fed a vitamin Bj 2 deficient diet (Beck, 1975 Herbert and Das, 1976) even though levels of serum B 2 methylmalonyl-CoA mutase activities and tissue coenzyme levels may be reduced. This may explain why with the exception of an early report on depressed complement fixing antibodies in vitamin deficient rats (Wertman and Sarandria, 1952) studies of vitamin B 2 deprivation in rat models have not demonstrated any effect on immune function. 

However, in some cases the reaction coordinate actually extends from the initial active-site selection into the protein, and the same-configuration solution is not adequate. One example appears in the study of Methylmalonyl-CoA mutase described below. Another drawback of a static optimization scheme is that it... 

Cobalamin-c disease remethylation of homocysteine to methionine also requires an activated form of vitamin B12. In the absence of normal B12 activation, homocystinuria results from a failure of normal vitamin B12 metabolism. Complementation analysis classifies defects in vitamin B12 metabolism into three groups cblC (most common), cblD and cblF. Most individuals become ill in the first few months or weeks of life with hypotonia, lethargy and growth failure. Optic atrophy and retinal changes can occur. Methylmalonate excretion is excessive, but less than in methylmalonyl-CoA mutase deficiency, and without ketoaciduria or metabolic acidosis. 

The fibroblasts do not convert cyanocobalamin or hydroxocobalamin to methylcobalamin or adenosyl-cobalamin, resulting in diminished activity of both N5-methyltetrahydrofolate homocysteine methyltransferase and methylmalonyl-CoA mutase. Supplementation with hydroxocobalamin rectifies the aberrant biochemistry. The precise nature of the underlying defect remains obscure. Diagnosis should be suspected in a child with homocystinuria, methylmalonic aciduria, megaloblastic anemia, hypomethioninemia and normal blood levels of folate and vitamin B12. A definitive diagnosis requires demonstration of these abnormalities in fibroblasts. Prenatal diagnosis is possible. 

The vitamin cobalamin (vitamin Bjj) is reduced and activated in the body to two forms, adeno-sylcobalamin, used by methylmalonyl CoA mutase, and methylcobalamin, formed from methyl-THF in the N-methyl THF-homocysteine methyltransferase reaction. These are the only two enzymes that use vitamin (other than the enzymes that reduce and add an adenosyl group to it). 

Propionyl-CoA is first carboxylated to form the d stereoisomer of methylmalonyl-CoA (Pig. 17—11) by propionyl-CoA carboxylase, which contains the cofactor biotin. In this enzymatic reaction, as in the pyruvate carboxylase reaction (see Pig. 16-16), C02 (or its hydrated ion, HCO ) is activated by attachment to biotin before its transfer to the substrate, in this case the propionate moiety. Formation of the carboxybiotin intermediate requires energy, which is provided by the cleavage of ATP to ADP and Pi- The D-methylmalonyl-CoA thus formed is enzymatically epimerized to its l stereoisomer by methylmalonyl-CoA epimerase (Pig. 17-11). The L-methylmal onyl -CoA then undergoes an intramolecular rearrangement to form succinyl-CoA, which can enter the citric acid cycle. This rearrangement is catalyzed by methylmalonyl-CoA mutase, which requires as its coenzyme 5 -deoxyadenosyl-cobalamin, or coenzyme Bi2, which is derived from vitamin B12 (cobalamin). Box 17—2 describes the role of coenzyme B12 in this remarkable exchange reaction. 

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.

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). 

S-Methylmalonyl-CoA mutase (EC 5.4.99.2) is a deoxyadenoxyladen-osylcobalamin-dependent enzyme of mitochondria required to catalyze the conversion of methylmalonyl-CoA to succinyl-CoA. A decrease in the activity of methylmalonyl-CoA mutase leads to the urinary excretion of large amounts of methylmalonic acid (C22). The biochemical lesion may be at the mutase level due to an abnormality of apoenzyme protein or an inability to elaborate the required coenzyme form of vitamin B12> i.e., adenosyl-cobalamin. In rare cases the abnormality may be due to an inability to convert the d form of methylmalonyl-CoA mutase to the l form as a result of a defective racemase (EC 5.1.99.1) (Kll). In patients, the nature of the abnormality can be determined by tissue culture studies (D13) and by clinical trial, since patients with a defect in adenosylcobalamin production will show clinical improvement when treated with very large doses of vitamin B12 (Mil). 

Thoma NH, Meier TW, Evans PR, Leadlay PF (1998) Stabilization of radical intermediates by an active-site tyrosine residue in methylmalonyl-CoA mutase. Biochemistry 37 14386-14393... 

The steric course of the methylmalonyl-CoA mutase reaction, as it affects the C-2 of methylmalonyl-CoA, can be elucidated by determining the absolute configuration of the substrate and of a suitably labelled product. The former problem was solved by two groups [30,31]. Briefly, the (25) configuration of the epimeric product obtained from enzymic carboxylation of propionyl-CoA was established and the (2R) configuration of the mutase-active methylmalonyl-CoA followed per ex-clusionem. 

Methylmalonyl CoA arises directly as an intermediate in the catabolism of valine, and is formed by the carboxylation of propionyl CoA arising in the catabolism of isoleucine, cholesterol, and fatty acids with an odd number of carbon atoms. Normally, as shown in Figure 10.13, it undergoes an adeno-sylcobalamin-dependentrearrangementto succinyl CoA, catalyzed by methylmalonyl CoA mutase. In vitamin B12 deficiency, the activity of this enzyme is greatly reduced, although there is induction of the apoenzyme to some 1.5- to 5-fold above that seen in control animals. 

Both methylmalonyl-CoA mutase and glutamate mutase share strikingly similar global folds (Figure 8), even though sequence similarity is limited to the small a/ 3 domain and their quaternary structures are quite different. The icatalytici domains of both enzymes take the form of a ( a/p)s TIM-barrel the Ca atoms of the two structures can be superimposed with an r.m.s. deviation of only 2 (Reitzer et al., 1999). However, the active site residues of these enzymes (other than those involved in binding the lower face of the coenzyme) do not seem to be conserved and the substrates are bound very differently. 

FIGURE 22. Detail of the active site of methylmalonyl-CoA mutase showing the interactions of Tyr-89 with substrate and coenzyme. 

Figure 22.16. Active Site of Methylmalonyl CoA Mutase. The arrangement of substrate and coenzyme in the active site facilitates the cleavage of the cobalt-carbon bond and the subsequent abstraction of a hydrogen atom from the substrate. 

The assay for serum Bn levels is a direct lest, as it measures the concentration of the vitamin itself. The assay of MM A levels is a functional test of B12 status, as it measures a compound whose metabolism is dependent on the correct functioning of vitamin Bn- The results of the MMA test reflect the activity of methylmalonyl-CoA mutase in the liver It is thought that the functional test is more valuable than the direct test. Serum vitamin Bij levels may not reflect the functioning of the B j-requiring enzymes of the cell. Serum Bii levels may sometimes be withm the normal range despite an increased excretion of MMA. Normal scrum Bt2 values range from 0.2 to 1,0 ng/ml. Normal serum MMA levels range from 20 to 75 ng/ml, and normal urinary MMA levels from 0,6 to 3-0 pg/ml. 

Figure 47-SO The major metabolic pathways for the use of ammonia by the hepatocyte. Solid bars indicate the sites of primary enzyme defects in various metabolic disorders associated with hyperammonemia /) carbamyl phosphate synthetase I, (2) ornithine transcarbamylase, (3) argininosuccinate synthetase, (4) argininosuccinate lyase, (5) arginase, (6) mitochondrial ornithine transport, (7) propionyi CoA carboxylase, (fi) methylmalonyl CoA mutase, (9) L-lysine dehydrogenase, and (10) N-acetyl glutamine synthetase. Dotted lines indicate the site of pathway activation (+) or inhibition ( ). (From Flannery OB, Hsia YE, Wolf 6. Current status of /lyperommofiemjo syndromes. Hepatology 1982 2 495-506,)...

---