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In Vitro Reaction of 3- Phenylpropionyl-Coa with Various Acyl-Coa Dehydrogenases

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In Vitro Reaction of 3- Phenylpropionyl-Coa with Various Acyl-Coa Dehydrogenases 003 1-3998/90/2705-050 l$02.00/0 PEDIATRIC RESEARCH Vol. 27, No. 5, 1990 Copyright Q 1990 International Pediatric Research Foundation. Inc. Prinled in U.S. A. The Enzymatic Basis for the Dehydrogenation of 3-Phenylpropionic Acid: In Vitro Reaction of 3- Phenylpropionyl-CoA with Various Acyl-CoA Dehydrogenases PIER0 RINALDO, JOHN J. O'SHEA, ROY D. WELCH, AND KAY TANAKA Yale University School ofMedicine, Department of Human Genetics, New Haven, Connecticut 06510 ABSTRACT. 3-Phenylpropionic acid is an end-product of 2-meBCAD, 2-methyl branched chain acyl-CoA dehydro- the bacterial degradation of unabsorbed phenylalanine in genase the intestinal lumen. As CoA ester, this metabolite has m/z, mass to charge ratio been considered to be a specific substrate for medium chain PMS, phenazine methosnlfate acyl-CoA dehydrogenase (MCAD). Its glycine-conjugate, PPA, 3-phenylpropionic acid 3-phenylpropionylglycine, has now been established as a PP-CoA, 3-phenylpropionyl-CoA pathognomonic marker in urine from patients affected with PPG, 3-phenylpropionylglycine MCAD deficiency. However, no systematic studies to eval- SCAD, short chain acyl-CoA dehydrogenase uate the reactivity of 3-phenylpropionyl-CoA with other SIM, selected ion monitoring known acyl-CoA dehydrogenases have so far been carried TMS, trimethylsilyl (derivative) out to establish the specificity of this substrate for MCAD. We studied the in vitro reactivity of 3-phenylpropionyl- CoA with five rat and human liver acyl-CoA dehydrogen- ases using purified preparations. We demonstrated that MCAD effectively dehydrogenated 3-phenylpropionyl- Phenylalanine metabolites have been investigated by gas chro- CoA, and that no other acyl-CoA dehydrogenase exhibited matography and GC/MS over the span of more than 20 y (1-3) any significant activity with this substrate. In the steady because of their significance in the biochemical characterization state condition, the Km of 3-phenylpropionyl-CoA for hu- of phenylketonuria and other types of inherited disorders of man MCAD was 50 wM. Gas chromatography/mass spec- phenylalanine hydroxylation (4). Among them, PPA is the end- trometry analysis of the assay mixture identified trans- product of a minor alternative pathway: the bacterial degradation cinnamoyl-CoA as the product of the reaction. Fnrther- of unabsorbed phenylalanine in the intestinal lumen (5-7). Lac- more, we showed by determination of the reaction products tobacillus pastorianus var. quinicus (a), Clostridium dlfficile (9), using gas chromatography/mass spectrometry selected ion and Peptostreptococcus anaerobius (10) represent some of the monitoring that, in absence of the primary electron accep- anaerobic bacterial strains that showed large amounts of PPA in tor, 3-phenylpropionyl-CoA was slowly but significantly ether-extracts from broth cultures analyzed by GC/MS (1 1) or dehydrogenated by MCAD under aerobic conditions. These liquid chromatography analysis (12). As summarized in Figure data suggest that MCAD may oxidize 3-phenylpropionyl- 1, several enzymatic steps are involved in this secondary pathway, CoA in vivo using an alternative electron acceptor, to where the mechanism of phenyllactic acid transformation to produce trans-cinnamoyl-CoA. This mechanism provides trans-cinnamic acid still requires direct experimental confirma- an explanation for the normal 3-phenylpropionylglycine tion. excretion observed in urine from patients affected with Unlike other phenylalanine metabolites (5-7), PPA is not glutaric aciduria type I1 and ethylmalonic/adipic aciduria. ordinarily detected as a free acid (1 3) in either phenylketonuric (Pediatr Res 27: 501-507,1990) patients (1-3) or normal subjects, even after a phenylpropionate loading test (14). However, it has been shown that homologous Abbreviations p-hydroxy intermediates were produced in tyrosine-loaded rats and rabbits along a parallel pathway (1 5), suggesting a different CI, chemical ionization endogenous metabolism in other mammalian species. Under CoA, coenzyme A normal circumstances, PPA absorbed from the human intestine DCIP, 2,6-dichlorophenol-indophenol circulates through the portal system into the liver and diffuses EI, electron impact ionization into the hepatocytes, where it is activated to the CoA ester. PP- ETF, electron transfer flavoprotein CoA is transformed to benzoyl-CoA (16), presumably via a 6- FAD, flavin adenine dinucleotide oxidation of its aliphatic chain that is initiated by an acyl-CoA GC/MS, gas chromatography/mass spectrometry dehydrogenase. PP-CoA is then converted to its glycine-conju- IVD, isovaleryl-CoA dehydrogenase gate (hippuric acid) by a mitochondria1 enzyme, benzoyl- LCAD, long chain acyl-CoA dehydrogenase CoA:glycine N-acyltransferase (17), and finally excreted in urine MCAD, medium chain acyl-CoA dehydrogenase (7). Received October 25, 1989; accepted December 19, 1989. The in vivo metabolism of PPA is now receiving considerable Correspondence and reprint requests: Piero Rinaldo, M.D., Yale University attention in a different context in the study of inborn errors of School of Medicine, Department of Human Genetics, 333 Cedar Street, P.O. Box metabolism. Its glycine conjugate, PPG, has proven to be a 3333, New Haven, CT 065 10-8005. Supported by Grants DK-299 1 1, DK- 17453, and DK-38 154 from NIH Grant pathognomonic metabolite for the diagnosis of MCAD defi- 1-378 from March of Dimes. ciency (18). The disease is an autosomal recessive defect of 50 502 RINALDO ET AL mitochondrial P-oxidation of fatty acids (19). PPG was originally enzyme from the substrate are transferred to coenzyme Q in the identified by GC/MS in urine from a case diagnosed as Reye's- main mitochondrial electron transport chain via two obligate like syndrome (20), and, 2 y later, was detected in urine from electron acceptors, ETF and ETF-ubiquinone oxidoreductase eight proven MCAD-deficient patients (14, 21). The specificity (29). It has been observed that the substrate specificities of acyl- of PPG for the diagnosis of MCAD deficiency has been recently CoA dehydrogenases overlap to some degree (26). So far, no confirmed by us in 21 MCAD-deficient patients including 17 evidence has ever been presented that PP-CoA is dehydrogenated cases who were asymptomatic and untreated at the time of the exclusively by MCAD and not by other mitochondrial acyl-CoA study (18). The hypothesis that PPA is a product of bacterial dehydrogenases. Thus, the specificity of PPG excretion for the metabolism has been corroborated by our observation that, diagnosis of MCAD deficiency has not yet been thoroughly among three diagnostic acylglycines, only PPG excretion was not confirmed by excluding other enzyme defects that could impair increased in urine from a MCAD-deficient newborn, in agree- PPA oxidation and cause PPG accumulation in body fluids. ment with the gradual bacteria colonization of the intestinal For these reasons, we synthesized the phenylpropionyl-CoA mucosa (Bennett MJ, Coates PM, Hale DE, Millington DS, ester and tested it in vitro as a substrate for five mitochondrial Pollitt RJ, Rinaldo P, Roe CR, Tanaka K, unpublished data). acyl-CoA dehydrogenases purified from rat liver (26-28) and In view of these recent development in the study of MCAD two from human liver (32). Also, a study of the reaction mech- deficiency and other inborn metabolic disorders, it is important anism was carried out by GC/MS analysis of the reaction prod- to ascertain if PP-CoA is metabolized exclusively via MCAD to ucts. Octanoyl-CoA, the optimal substrate of MCAD, and PP- provide the biochemical basis for the use of PPG as a diagnostic CoA were allowed to react with human MCAD under various marker for MCAD-deficiency. Previous in vivo (16) and in vitro assay conditions. These experiments were carried out to confirm (22, 23) experiments have shown that the shortening of the the identity of the trans-2-enoyl-CoA products and to explore aliphatic chain of w-phenyl fatty acids by two carbon atoms is the mechanism(s) for the normal PPG urinary excretion in catalyzed by the enzymes of mitochondrial and peroxisomal fatty patients affected with ETF or ETF-ubiquinone oxidoreductase acid P-oxidation (23). In an extensive study of the mechanism of deficiencies, commonly referred to as glutaric aciduria type I1 or the flavin reduction step in the acyl-CoA dehydrogenation path- ethylmalonic/adipic aciduria (29). way, Murfin (22) investigated the dehydrogenation of PP-CoA by mitochondrial octanoyl-CoA dehydrogenase, that was pre- MATERIALS AND METHODS pared from beef liver. However, it should be noted that there are six known acyl-CoA dehydrogenases (24, 25). Three of them, Materials. CoA lithium salt was obtained from Pharmacia SCAD, MCAD, and LCAD, catalyze the first reaction of the P- (Milwaukee, WI), FAD and DCIP were purchased from Sigma oxidation cycle in the catabolism of fatty acids with varying Chemical Co. (St. Louis, MO), PMS was from Eastman (Roch- chain length (26). Two others IVD (27) and 2-meBCAD (28), ester, NY). Hydrocinnamic, octanoic, trans-2-octenoic, trans- catalyze the third reaction in the leucine and isoleucine/valine cinnamic, and 4-phenylbutyric acids were purchased from Ald- pathways, respectively. Glutaryl-CoA dehydrogenase catalyzes rich (Milwaukee, WI). D,~-Octanoicacid was obtained from the oxidative decarboxylation of glutaryl-CoA, an intermediate Merck, Sharp and Dohme (Montreal, Canada). N,O-bis (tri- of L-lysine metabolism, yielding as final products crotonyl-CoA methylsily1)-acetamide in pyridine and 1.0 mL Reactivials were and carbon dioxide (29). The first five of these enzymes belong from Pierce Chemical Co. (Rockford, IL). Diazomethane was to the acyl-CoA dehydrogenase family (30) and share similar generated by the method of Schlenk and Gellerman (33) with molecular properties and reaction mechanisms (31). All acyl- minor modifications (34). CoA dehydrogenases remove one hydrogen each from the 0- and Synthesis ofphenylpropionyl-CoA. PP-CoA was prepared from a-positions of an acyl-CoA ester. Electrons accepted by the free CoA and hydrocinnamic acid via the mixed anhydride synthesis (35, 36). PP-CoA was purified by ascending paper chromatography using ethanol/O.
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