An enzyme is extracted from the red peel of Amanita muscaria which cleaves the C(2)-C(3) and the C(4)-C(5) bond of the aromatic ring of L-dopa (1) to form a mixture of 4,5-secodopa ( = salt of 6-amino-2-hydroxy-4-(2'-oxoethylidene)hept-2-enedioic acid; 2) and 2,3-secodopa ( = salt of 7-amino-5-formyl-2-hydroxyocta-2,4-dienedioic acid; 3), two hitherto hypothetical biosynthetic intermediates (see Scheme). Though isolation of these products has not been possible, structural evidence is inferred from reaction products, kinetics, and spectroscopical characteristics in comparison with known compounds. Secodopas 2 and 3 are characterized in dilute solution by HPLC and UVjVIS spectroscopy (anions: I,,, 424 and 414 nm, resp., E~~~ = 25 500; on acidification, shift to 380 and 372 nm, resp.). They cyclize without enzyme catalysis, optimally at pH 4.5-5; 3 produces muscaflavin (5) and 2 betalamic acid (4). The products are identified by direct comparison with authentic samples in HPLC, by 'H-NMR of 5, and by condensation of 4 with L-proline to form the well known betalain indicaxanthin (7). The enzymatic conversion of L-dopa (1) via 2 to betalamic acid (4; (5')) and its condensation with L-proline leads to pure natural indicaxanthin (7; (2S, 1 1s)); correspondingly, the enzymatic conversion of o-dopa to (R)-betalamic acid and its condensation with L-proline produces isoindicaxanthin ((2S,IlR)) which is unknown in nature. Particularly relevant is the fact that the same enzyme cleaves pyrocatechol to produce a solution of the enolate form of the known 2-hydroxy-6-oxohexa-2,4-dienoate (secopyrocatechol; 9; see Fig. 5 ) . Dissociation constants of the corresponding enolic functions in the cleavage products are determined by spectrometric titration and compared to those of known systems.

390J. Am. Chem. SOC. with 0.37 unit of transcarboxylase in 20 p L containing KP, (250 mM, pH 7.0) and DTT (IO mM) at 25 OC. After 1 min the incubation was centrifuged through two spun-dry DE52 ( I mL wet) beds in sequence in the cold within 10 min. The 14C radioactivity and enzyme activity of the second filtrate were compared. This ratio was determined as a function of the MMCoA concentration used to label the enzyme: 0.05,0.1, and 0.25 mM. The limit, determined graphically, was 1280 cpm/unit of activity; i.e. 0.275 nmol of biotin could be carboxylated by MMCoA per unit of activity. This value is -40% greater than the highest value reported by Wood et a1.I0 on the basis of the counts of labeled biotin 1993,115, 390-396 incorporated and the highest enzyme activity that was obtained in the same assay. The difference can be attributed to loss during storage of the assay rate, which depends on the coordinated function of the two half-reactions, without loss of capacity for the MMCoA/propionyl CoA half-reaction.* Acknowledgment.Abstract: Prochiral selectivity of various hydrolytic enzymes (lipases and proteases) in organic solvents was investigated in transformations involving a 2-substituted 1,3-propanedioI or its diester. In two instances, a significant dependence of enzyme prochiral selectivity on the solvent was found: transesterification of diol 1 with vinyl butyrate catalyzed by Aspergillus oryzae protease in anhydrous solvents and hydrolysis of diester 3 catalyzed by Pseudomonas sp. lipase in hydrated organic solvents (monoester 2 was a product in both reactions). The latter process, where the p r o 4 selectivity of the enzyme varied from around 3 in some solvents to greater than 30 in others, was examined in more detail. A mechanistic model was proposed that predicted an inverse correlation between lipase's prochiral selectivity and solvent hydrophobicity, as well as particular effects of substrate structure variation and an additive on the prochiral selectivity; all these predictions were confirmed experimentally. Subtilisin Carlsberg lacked appreciable prochiral selectivity in either transesterification or hydrolysis reactions regardless of the solvent; this was rationalized by means of interactive computer modeling based on the X-ray crystal structure of this serine protease.

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