Stanley D. Young

Oxygen-18 isotopic carbon-13 NMR shift as proof that bifunctional peptidylglycine .alpha.-amidating enzyme is a monooxygenase

: The biosynthesis of C-terminal alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine alpha-amidating enzyme (alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. Using bifunctional type A rat alpha-AE, we have shown that O2 is the source of the alpha-carbonyl oxygen of pyruvate produced during the amidation of dansyl-Tyr-Val-[alpha-13C]-D-Ala, as demonstrated by the 18O isotopic shift in the 13C NMR spectrum of [alpha-13C]lactate generated from [alpha-13C]pyruvate in the presence of lactate dehydrogenase and NADH. In addition, one-to-one stoichiometries have been determined for glyoxylate formed/dansyl-Tyr-Val-Gly consumed, pyruvate formed/dansyl-Tyr-Val-D-Ala consumed, dansyl-Tyr-Val-NH2 formed/ascorbate oxidized, and dansyl-Tyr-Val-NH2 formed/O2 consumed. Quantitative coupling of NADH oxidation to dansyl-Tyr-Val-NH2 production using Neurospora crassa semidehydroascorbate reductase showed that two one-electron reductions by ascorbate occurred per alpha-AE turnover. The stoichiometry of approximately 1.0 dansyl-Tyr-Val-NH2 produced/ascorbate oxidized observed in the absence of a semidehydroascorbate trap resulted from the disproportionation of two semidehydroascorbate molecules to ascorbate and dehydroascorbate.

Selective inactivation of the hydroxylase activity of bifunctional rat peptidylglycine α-amidating enzyme

Conversion of dansyl-Tyr-Val-Gly to dansyl-Tyr-Val-NH2 by recombinant type A rat 75-kDa peptidylglycine alpha-amidating enzyme (alpha-AE) is inactivated by ascorbate, dehydroascorbate, and hydrogen peroxide in a time- and concentration-dependent manner. Both ascorbate- and dehydroascorbate-mediated inactivation are saturable with apparent kinact/Kinact values of 1.7 and 0.23 s-1 M-1, respectively. Hydrogen peroxide-mediated inactivation is not saturable with a second-order rate constant of 50 s-1 M-1. Peptidyl-Gly substrates, EDTA, and H2O2 scavengers protect against ascorbate-mediated inactivation while EDTA and semidehydroascorbate scavengers protect against dehydroascorbate-mediated inactivation. Under similar conditions, ascorbate, dehydroascorbate, and H2O2 have no effect on the alpha-AE-catalyzed conversion of dansyl-Tyr-Val-alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 which is consistent with the hypothesis that the 75-kDa enzyme consists of distinct peptidyl-Gly hydroxylase and peptidyl-alpha-hydroxyglycine lyase active sites.

Methods for the carboxyl-terminal fluorescent labeling of peptides using solid phase peptide synthesis

Abstract Two general methods for labeling synthetic peptides with a 5-dimethylamino-1-napthalenesulfonyl (dansyl) group at the C-terminal residue using solid phase peptide synthesis (SPPS) are described. Dansylated peptides are ideal substrates for fluorometric proteolytic enzyme assays.

Rapid fluorimetric assay for the detection of the peptidyl α-amdating enzyme intermediate using high-performance liquid chromatography

Peptidylglycine alpha-amidating enzyme catalyzes the conversion of glycine-extended peptides to their corresponding amidated peptides via a stable alpha-hydroxyglycine intermediate. Using a new rapid fluorimetric reversed-phase high-performance liquid chromatographic assay, we have demonstrated that the substrate and product of the amidation reaction, as well as both stereoisomers of the alpha-hydroxyglycine intermediate, can be separated and detected in quantities as low as 1 pmol. The method is highly reproducible and requires less than 11 min for separation and quantification.

The Enzymology of Peptide Amidation

Since the 1950’s, it has been recognized that most peptide hormones contain a C-terminal amide3–6. In the last 40 years, over 100 amidated peptides have been identified7,8 and structure-activity relationships have shown that the C-terminal amide is key to the activity elicited by most amidated peptides9–12. The amide moiety arises by the post-translational, oxidative cleavage of a C-terminal glycine-extended prohormone (peptidyl-Gly) at the a-carbon of the glycine in a reaction which requires a reducing equivalent, copper, and O2 13,14. Peptidylglycine α-amidating enzyme (α-AE, EC 1.14.17.3)15,16 is the enzyme that catalyzes this reaction in vivo. In this review, we will focus on the chemical and enzymological aspects of peptide amidation. For more comprehensive treatments of the subject, two recent reviews are recommended8,17.

A four step synthesis from α-pinene of 7,7-dimethylbicyclo[4.1.1]octa-2,4-diene

α-Pinen (I) wird der Ozonolyse zum Keto-aldehyd (II) unterworfen und dieser mit Piperidin/ Eisessig zu (IIIa) cyclisiert.

High Performance Liquid Chromatography of Phenylthiohydantoin and Phenylthiocarbamyl Amino Acids

Microsequence analysis of polypeptides by the Edman procedure has become a standard and routine method for the protein chemist (1,2). This cyclic stepwise process is initiated by the reaction of phenylisothiocyanate (PITC) with the amino terminus of a polypeptide. Following this coupling step, the derivatized amino-terminal residue is selectively cleaved from the polypeptide and converted to a phenylthiohydantoin (PTH) amino acid. Various reverse-phase high performance liquid chromatography (HPLC) systems have been reported for the identification and quantitation of the resulting PTH amino acids (3–8). Recently, methods have been described which also use PITC derivatization for amino acid analysis (9,10). This procedure involves the reaction of PITC with amino acids to form phenylthiocarbamyl (PTC) derivatives and their subsequent identification and quantitation by reverse-phase HPLC.