Adenylosuccinate synthetase from maize. Purification, properties, and mechanism of inhibition by 5'-phosphohydantocidin.
Adenylosuccinate synthetase (AdSS) is the site of action hydantocidin, a potent microbial phytotoxin. A kinetic analysis of the mode of inhibition of a plant adenylosuccinate synthetase by the active metabolite 5'-phosphohydantocidin (5'-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 microM, respectively. Inhibition of AdSS by 5'-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'-PH was competitive with IMP, with an apparent Ki of 22 nM. Apparently, 5'-PH inhibits the enzyme by binding to the IMP site and forming a tight, dead-end complex.
Walters
E W
EW
Novartis Crop Protection, Research Division, Palo Alto, California 94304-1104, USA.
Lee
S F
SF
Niderman
T
T
Bernasconi
P
P
Subramanian
M V
MV
Siehl
D L
DL
D000265:Adenylosuccinate Synthase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D006827:Hydantoins / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010428:Pentosephosphates / Q000494:pharmacology; D010940:Plant Proteins / Q000302:isolation & purification*; D017421:Sequence Analysis; D003313:Zea mays / Q000201:enzymology*
C106827:5'-phosphohydantocidin; D006827:Hydantoins; D010428:Pentosephosphates; D010940:Plant Proteins; D000265:Adenylosuccinate Synthase