Search PubMed⌕ Search

Biomedical subjects

P Marcotte

Publications and source records attributed to P Marcotte.

25 records · Page 2Linked to original sources
PubMed1978

Sequence of reactions which follows enzymatic oxidation of propargylglycine.

The nonenzymatic reactions which follow enzymatic oxidation of the gamma-delta acetylenic amino acid propargylglycine (2-amino-4-pentynoate) have been studied. The product which accumulates in solution has been identified as 2-amino-4-hydroxy-2,4-pentadienoate gamma-lactone, formed by intramolecular attack of the carboxylate anion on the electrophilic fourth carbon of 2-iminium-3,4-pentadienoate. This previously unknown substance was characterized by its reactions in acid and base and by its nuclear magnetic resonance spectrum. The lactone is preceded in the pathway by 2-amino-2-penten-4-ynoate, a transient electron-rich species which binds tightly to D-amino-acid oxidase and induces a charge-transfer complex with the electron-deficient bound flavin coenzyme. The aminediene lactone is converted by base treatment to 2-amino-4-keto-2-pentenoate, which is also a strong inhibitor of D-amino-acid oxidase and induces a charge-transfer complex.

Alkynes↗
PubMed1978

Sequence of reactions which follows enzymatic oxidation of allylglycine.

The pathway following flavoprotein-catalyzed oxidation of allylglycine (2-amino-4-pentenoate) has been studied and found to be dependent on the incubation conditions. In N-2-hydroxyethyl-N'-2-ethanesulfonic acid (Hepes) buffer, the oxidation product 2-iminium-4-pentenoate predominantly reacts to form 2-amino-2,4-pentadienoate, a strong noncovalent inhibitor of D-amino-acid oxidase. However, in pyrophosphate buffer, the more rapid reaction is hydrolysis to form 2-keto-4-pentenoate, which has been found to be a substrate for L-lactic dehydrogenase. 2-Keto-4-pentenoate is in rapid equilibrium with 2-hydroxy-2,4-pentadienoate, which is also a strong noncovalent inhibitor of D-amino-acid oxidase. In both systems, these metastable intermediates react in subsequent slower steps to yield trans-2-keto-3-pentenoate, which accumulates in the incubation. Syntheses of trans-2-amino- and trans-2-keto-3-pentenoate are described. Comparisons between the reactivities of acetylenic and olefinic species have been made based on the differences between this pathway and that following oxidation of propargylglycine [Marcotte, P., and Walsh, C. (1978), Biochemistry 17 (preceding paper in this issue)].

Alcohol Oxidoreductases↗
PubMed1978

Properties of D-amino acid oxidase covalently modified upon its oxidation of D-propargylglycine.

Upon oxidation of D-propargylglycine by D-amino acid oxidase, the enzyme is converted by covalent alkylation to catalytic species with different properties from those of native enzyme. At least five distinct modified enzyme species are present in the preparation, as determined by gel electro-focusing. Individual characterization of the components has not yet been attempted. The combined kinetic and spectral properties of the preparation have been studied. The modified enzymes have a marked preference for hydrophobic amino acids: the rates of oxidation decrease in the series D-phenylalanine, D-methionine, D-norleucine, D-norvaline, D-alpha-aminobutyrate, D-alanine. In addition, the observed Kms of the amino acids are increased, especially those of the smaller substrates (D-alanine and D-alpha-aminobutyrate). A primary kinetic isotope effect is observed upon oxidation of amino acids by the modified enzymes, evidence that this catalysis exhibits a different rate-determining step from catalysis by native enzyme. The modified apoenzyme exhibits intense absorbance at 318--320 nm, not present in native enzyme. This chromophore can be partially (75%) removed by treatment of the modified enzyme with hydrazine. However, the activity of native enzyme is not substantially restored by this process, suggesting the existence of superficial alkylations in addition to the modification responsible for the observed changes in kinetic parameters.

Alkynes↗
PubMed1976

Vinylglycine and proparglyglycine: complementary suicide substrates for L-amino acid oxidase and D-amino acid oxidase.

Proparglyglycine (2-amino-4-pentynoate) and vinylglycine (2-amino-3-butenoate) have been examined as substrates and possible inactivators of two flavo enzymes, D-amino acid oxidase from pig kidney and L-amino acid oxidase from Crotalus adamanteus venom. Vinylglycine is rapidly oxidized by both enzymes but only L-amino acid oxidase is inactivated under assay conditions. The loss of activity probably involves covalent modification of an active site residue rather than the flavin adenine dinucleotide coenzyme and occurs once every 20000 turnovers. We have confirmed the recent observation (Horiike, K, Hishina, Y., Miyake, Y., and Yamano, T. (1975) J, Biochem. (Tokyo), 78, 57) that D-proparglglycine is oxidized with a time-dependent loss of activity by D-amino acid oxidase and have examined some mechanistic aspects of this inactivation, The extent of residual oxidase activity, insensitive to further inactivation, is about 2%, at which point 1.7 labels/subunit have been introduced with propargly[2-14C]glycine as substrate. L-Proparglyclycine is a substrate but not an inactivator of L-amino acid oxidase and the product ahat accumulats in the nonnucleophilic N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer is acetopyruvate. In the presence of butylamine HCl, a species with lambdaman 317 nm (epsilon = 15 000) accumulates that may be a conjugated eneamine adduct. The same species accumulates from D-amino acid oxidase oxidation of D-propargylglycine prior to inactivation; the inactivated apo D-amino acid oxidase has a new peak at 317 nm that is probably a similar eneamine. A likely inactivating species is 2-keto-3,4-pentadienoate arising from facile rearrangement of the expected initial product 2-keto 4 pentynoate. Vinylglycine and proparglyglycine show inactivation specificity, then, for L-and D-amino acid oxidase, respectively.

Alkynes↗