PLANT PHYSIOLOGY , Vol 114, Issue 2 549-555, Copyright © 1997 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Adenylosuccinate Synthetase from Maize (Purification, Properties, and Mechanism of Inhibition by 5[prime]-Phosphohydantocidin)
E. W. Walters, S. F. Lee, T. Niderman, P. Bernasconi, M. V. Subramanian and D. L. Siehl
Novartis Crop Protection, Research Division, 975 California Avenue, Palo Alto, California 94304-1104
Adenylosuccinate synthetase (AdSS) is the site of action of hydantocidin, a
potent microbial phytotoxin. A kinetic analysis of the mode of inhibition
of a plant adenylosuccinate synthetase by the active metabolite
5[prime]-phosphohydantocidin (5[prime]-PH) was the objective of the present
study. AdSS was purified 5800-fold from maize (Zea mays), to our knowledge
the first purification of the enzyme from a plant source. N-terminal
sequencing established the cleavage site of the previously published
deduced sequence of the initial transcript. The subunit molecular mass was
determined to be 48 kD and the isoelectric point was at pH 6.1. Values of
the Michaelis constant for the three substrates IMP, GTP, and aspartate
were 21, 16, and 335 [mu]M, respectively. Inhibition of AdSS by 5[prime]-PH
was measurably time-dependent. The trace of the inactivation curve could
not be altered by preincubating the enzyme and inhibitor in the absence of
substrates but could be linearized by preincubating the enzyme with
inhibitor, aspartate, GTP (or GDP), and inorganic phosphate. Inhibition of
AdSS by 5[prime]-PH was competitive with IMP, with an apparent Ki of 22 nM.
Apparently, 5[prime]-PH inhibits the enzyme by binding to the IMP site and
forming a tight, dead-end complex.
