Leucine enzymic oxidation

As indicated above, the reaction catalyzed by the general d- and l-amino acid oxidases has been represented by a dehydrogenation of an amino acid by a flavoenzyme to jneld reduced flavoenzyme and the corresponding imino acid [reactions (2) - - (4)]. Indirect support for the formation of the hypothetical imino acid has been provided by a number of studies which exclude a,/3-unsaturation in the course of the reaction. For example, it has been shown that (a) the four isomers of isoleucine are enzymically oxidized by the appropriate amino acid oxidase to the corresponding optically active a-keto-/3-methylvaleric acids (5, 6), (b) the l-isomers of /3-phenylserine are converted by L-amino acid oxidase to the respective isomers of mandelic acid (7), (c) the l- and D-isomers of a-aminophenylacetic acid, which have no /3-hydrogen atom, are attacked by the amino acid oxidases 8, 9), aod (d) on the oxidation of L-leucine in the presence of D,0 by L-amino acid oxidase, no deuterium is found in the isolated a-ketoisocaproic acid (10). More direct evidence for the formation of the a-imino acid as an intermediate has been provided by Pitt (10a) in studies on the oxidation of aromatic amino aci by ophto-n-amino acid oxidase in the presence of a tautomerase. 

Inherited defects in the enzymes of (3-oxidation and ketogenesis also lead to nonketotic hypoglycemia, coma, and fatty hver. Defects are known in long- and short-chain 3-hydroxyacyl-CoA dehydrogenase (deficiency of the long-chain enzyme may be a cause of acute fetty liver of pr nancy). 3-Ketoacyl-CoA thiolase and HMG-CoA lyase deficiency also affect the degradation of leucine, a ketogenic amino acid (Chapter 30). 

An entirely different property of subtilisin was affected by substituting leucine at the 222 location. Native BPN is extremely sensitive to the presence of oxidation agents, showing rapid inactivation when incubated in the presence of 0.3% H2O2 (Figure 4). The Leu-222 variant, in contrast, was found to be totally stable under the same oxidation conditions. The data clearly show that single amino acid alterations can have dramatic effects upon the activity of the enzyme. Similarly, other changes have been shown to affect catalytic properties, substrate specificities and thermostability (7,2,9). 

An elegant four-enzyme cascade process was described by Nakajima et al. [28] for the deracemization of an a-amino acid (Scheme 6.13). It involved amine oxidase-catalyzed, (i )-selective oxidation of the amino acid to afford the ammonium salt of the a-keto acid and the unreacted (S)-enantiomer of the substrate. The keto acid then undergoes reductive amination, catalyzed by leucine dehydrogenase, to afford the (S)-amino acid. NADH cofactor regeneration is achieved with formate/FDH. The overall process affords the (S)-enantiomer in 95% yield and 99% e.e. from racemic starting material, formate and molecular oxygen, and the help of three enzymes in concert. A fourth enzyme, catalase, is added to decompose the hydrogen peroxide formed in the first step which otherwise would have a detrimental effect on the enzymes. 

Figure 9-4. Metabolism of the branched-chain amino acids. The first two reactions, transamination and oxidative decarboxylation, are catalyzed by the same enzyme in all cases. Details are provided only for isoleucine. Further metabolism of isoleucine and valine follows a common pathway to propionyl CoA. Subsequent steps in the leucine degradative pathway diverge to yield acetoacetate. An intermediate in the pathway is 3-hydroxy-3-methylglutaryl CoA (HMG-CoA), which is a precursor for cytosolic cholesterol biosynthesis.

NAD+ serves as the oxidant. The reaction is catalyzed by a complex of enzymes whose molecular mass varies from 4 to 10 x 106, depending on the species and exact substrate.297 Separate oxoacid dehydrogenase systems are known for pyruvate,298-300 2-oxoglut-arate,301 and the 2-oxoacids with branched side chains derived metabolically from leucine, isoleucine, and... 

In a rare autosomal recessive condition (discovered in 1954) the urine and perspiration has a maple syrup odor/ High concentrations of the branched-chain 2-oxoacids formed by transamination of valine, leucine, and isoleucine are present, and the odor arises from decomposition products of these acids. The branched-chain amino acids as well as the related alcohols also accumulate in the blood and are found in the urine. The biochemical defect lies in the enzyme catalyzing oxidative decarboxylation of the oxoacids, as is indicated in Fig. 24-18. Insertions, deletions, and substitutions may be present in any of the subunits (Figs. 15-14,15-15). The disease which may affect one person in 200,000, is usually fatal in early childhood if untreated. Children suffer seizures, mental retardation, and coma. They may survive on a low-protein (gelatin) diet supplemented with essential amino acids, but treatment is difficult and a sudden relapse is apt to prove fatal. Some patients respond to administration of thiamin at 20 times the normal daily requirement. The branched-chain oxoacid dehydrogenase from some of these children shows a reduced affinity for the essential coenzyme thiamin diphosphate.d... 

In the degradation of isoleucine, (3 oxidation proceeds to completion in the normal way with generation of acetyl-CoA and propionyl-CoA. However, in the catabolism of leucine after the initial dehydrogenation in the (3-oxidation sequence, carbon dioxide is added using a biotin enzyme (Chapter 14). The double bond conjugated with the carbonyl of the thioester makes this carboxylation analogous to a standard (3-carboxylation reaction. Why add the extra C02 ... 

The driving force in the coupled enzyme process is independent of the equilibrium of the LeuDH catalyzed reaction because of the irreversibility of the NOX reaction. This is most favorable because the oxidation reaction of many NAD(P)+-dependent dehydrogenases is hampered by their equilibrium, which prefers the reduction reaction. By applying this system D-ferf-leucine was obtained with an excellent ee >99%. 

In mammals, there are only three vitamin B12 -dependent enzymes methionine synthetase, methylmalonyl CoA mutase, and leucine aminomutase. The enzymes use different coenzymes methionine synthetase uses methylcobal-amin, and cobalt undergoes oxidation during the reaction methylmalonyl CoA mutase and leucine aminomutase use adenosylcobalamin and catalyze the formation of a 5 -deoxyadenosyl radical as the catalytic intermediate. 

Branched-Chain Oxo-acid Decarboxylase and Maple Syrup Urine Disease The third oxo-add dehydrogenase catalyzes the oxidative decarboxylation of the branched-chain oxo-acids that arise from the transamination of the branched-chain amino acids, leucine, isoleuctne, emd vtdine. It has a similEU subunit composition to pyruvate and 2-oxoglutarate dehydrogenases, and the E3 subunit (dihydrolipoyl dehydrogenase) is the stune protein as in the other two multienzyme complexes. Genetic lack of this enzyme causes maple syrup urine disease, so-called because the bremched-chain oxo-acids that are excreted in the urine have a smell reminiscent of maple syrup. 

Vitamin Bu is unique among all the vitamins in that it is the largest and most complex and because it contains a metal ion. 1 hismetal ion is cobalt. Cobalt occurs in three oxidation states Co, Co, and Co. The medicinal forms of the vitamin are cyanocobalamin and hydnoxocobalamin. In cyanocobalamin, a molecule of Cyanide is complexed to the Co atom. Cyanocobalamin is readily converted in the body to the cofactor forms methylcobalamin and 5-deoxyadenosylcobalamin. Methylcobalamin contains cobalt in the Co slate, where it acts as a cofactor for methionine synthase. 5 Deoxyadenosylcobalamin, which contains cobalt in the Co state, is the cofactor for methylmalonyl-CoA mutase. Vitamin Btj is also a cofactor for leucine aminomutase, anen7yme used in leucine metabolism (Poston, 1984). This enzyme appears not to have a vital function in metabolism. No more... 

---