Elsevier

Phytochemistry

Volume 14, Issue 9, September 1975, Pages 1975-1982
Phytochemistry

Glutamine-dependent asparagine synthetase from Lupinus luteus

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Abstract

Asparagine synthetase (glutamine-hydrolyzing [l-aspartate: l-glutamine amido-ligase (AMP-forming), E.C. 6.3.5.4] was purified over 500-fold from cotyledon extracts of 1-week-old yellow lupin seedlings. The enzyme was labile and required protection by high levels of thiols; glycerol and the substrates also stabilized it. The reaction products were shown to be asparagine, AMP, PPi and glutamate. The limiting Km values were for aspartate 1·3 mM, for MgATP 0·14 mM and for glutamine 0·16 mM. Positive homotropic cooperativity was observed for MgATP only, and gel filtration studies indicated that the substrate-free enzyme (MW 160 000) associated to a dimer (MW 320 000 in the presence of MgCl2 and ATP. The purified enzyme, which had some glutaminase activity, catalyzed an aspartate- and glutamine-independent ATP-PPi exchange reaction at a rate 5–7-fold higher than the rate of asparagine synthesis. Initial velocity studies and exchange data indicated an overall ping-pong mechanism. Compared to similar enzymes isolated from mammalian tumor cells, the lupin enzyme appears to be unique with respect to MW, reaction mechanism and regulatory properties. The allosteric properties observed suggest an important role for this enzyme in the regulation of asparagine biosynthesis.

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