Purification and characterization of a novel 4-methyleneglutamine synthetase from germinated peanut cotyledons (Arachis hypogaea).
A newly detected amide synthetase, designated 4-methyleneglutamine synthetase, has been partially purified from extracts of 5- to 7-day germinated peanut cotyledons (Arachis hypogaea). Purification steps include fractionation with protamine sulfate and ammonium sulfate followed by column chromatography on Bio-Gel and DEAE-cellulose; synthetase purified over 300-fold is obtained. The enzyme has a molecular weight estimated to be approximately 250,000 and a broad pH optimum with maximal activity at approximately pH 7.5. Maximal rates of activity are obtained with NH+4 (Km = 3.7 mM) as the amide donor and the enzyme is highly specific for 4-methylene-L-glutamic acid (Km = 2.7 mM) as the amide acceptor. Product identification and stoichiometric studies establish the reaction catalyzed to be: 4-methyleneglutamic acid + NH4+ + ATP Mg2+----4-methyleneglutamine + AMP + PPi. PPi accumulates only when F- is added to inhibit pyrophosphatase activity present in synthetase preparations. This enzymatic activity is completely insensitive to the glutamine synthetase inhibitors, tabtoxinine-beta-lactam and F-, and is only partially inhibited by methionine sulfoximine. It is, however, inhibited by added pyrophosphate in the presence of F- as well as by certain divalent metal ions (other than Mg2+) including Hg2+, Ni2+, Mn2+, and Ca2+. All data obtained indicate that this newly detected synthetase is distinct from the well-known glutamine and asparagine synthetases.
D019732:Amide Synthases; D010367:Arachis / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D005459:Fluorides / Q000494:pharmacology; D005971:Glutamates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008025:Ligases / Q000302:isolation & purification*; D008345:Manganese / Q000494:pharmacology; D008628:Mercury / Q000494:pharmacology; D008970:Molecular Weight; D009532:Nickel / Q000494:pharmacology; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors; D013379:Substrate Specificity
D005971:Glutamates; D008345:Manganese; C030977:4-methyleneglutamic acid; D009532:Nickel; D011755:Pyrophosphatases; D008025:Ligases; D019732:Amide Synthases; C037658:4-methyleneglutamine synthetase; D008628:Mercury; D005459:Fluorides; D002118:Calcium