ADENYLOSUCCINATE SYNTHETASE FROM MAIZE - PURIFICATION, PROPERTIES, AND MECHANISM OF INHIBITION BY 5'-PHOSPHOHYDANTOCIDIN

Adenylosuccinate synthetase (AdSS) is the site of action of hydantocid in, a potent microbial phytotoxin. A kinetic analysis of the mode of i nhibition of a plant adenylosuccinate synthetase by the active metabol ite 5'-phosphohydantocidin (5'-PH) was the objective of the present st udy. AdSS was purified 5800-fold from maize (Zea mays), to our knowled ge the first purification of the enzyme from a plant source. N-termina l sequencing established the cleavage site of the previously published deduced sequence of the initial transcript. The subunit molecular mas s 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, an d aspartate were 21, 16, and 335 mu M, respectively. Inhibition of AdS S by 5'-PH was measurably time-dependent. The trace of the inactivatio n 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 pho sphate. Inhibition of AdSS by 5'-PH was competitive with IMP, with an apparent K-i of 22 nM. Apparently, 5'-PH inhibits the enzyme by bindin g to the IMP site and forming a tight, dead-end complex.