Lysine-insensitive aspartate kinase in two threonine-overproducing mutants of maize |
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Authors: | Stanton B Dotson David A Frisch David A Somers Burle G Gengenbach |
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Institution: | (1) Department of Agronomy and Plant Genetics, and Plant Molecular Genetics Institute, University of Minnesota, 55108 St. Paul, MN, USA;(2) Present address: Monsanto Company, 700 Chesterfield Village, Parkway, 63198 Chesterfield, MO, USA |
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Abstract: | Aspartate kinase (AK; EC 2.7.2.A) catalyzes the first reaction in the biosynthesis pathway for aspartate-derived amino acids
in plants. Aspartate kinase was purified from wildtype and two maize (Zea mays L.) genotypes carrying unlinked dominant mutations,Ask LT19 andAsk2 -LT20, that conferred overproduction of threonine, lysine, methionine and isoleucine. The objective of this investigation was to
characterize the AKs from mutant and wildtype plants to determine their role in regulating the synthesis of aspartate-derived
amino acids in maize. Kernels of the homozygousAsk2 mutant exhibited 174-, 10-, 13- and 2-fold increases in, in this sequence, free threonine, lysine, methionine and isoleucine,
compared to wildtype. In wildtype maize, AK was allosterically feedback-inhibited by lysine with 10 μMl-lysine required for 50% inhibition. In contrast, AK purified from the isogenic heterozygousAsk and homozygousAsk2 mutants required 25 and 760 μM lysine for 50% inhibition, respectively, indicating thatAsk andAsk2 were separate structural loci for lysine-regulated AK subunits in maize. Further characterization of purified AK from the
homozygous mutantAsk2 line indicated altered substrate and lysine inhibition kinetics. The apparent Hill coefficient was 0.7 for the mutantAsk2 AK compared with 1.6 for the wildtype enzyme, indicating that the mutant allele conferred the loss of a lysinebinding site
to the mutant AK. Lysine appeared to be a linear noncompetitive inhibitor ofAsk2 AK with respect to MgATP and an uncompetitive inhibitor with respect to aspartate compared to S-parabolic, I parabolic noncompetitive
inhibition of wildtype AK. Reduced lysine sensitivity of theAsk2 gene product appeared to reduce the lysine inhibition of all of the AK activity detected in homozygousAsk2 plants, indicating that maize AK is a heteromeric enzyme consisting of the two lysine-sensitive polypeptides derived from
theAsk andAsk2 structural genes.
Scientific paper No. 17419, Minnesota Agricultural Experiment Station projects No. 0302-4813-56 and No. 0302-4818-32
This research was supported in part by the U.S. Depatment of Agriculture Competitive Research Grants Office grant 86-CRCR-1-2019.
The authors are grateful to Charles Grissom for providing the computer programs in an IBM-PC format. |
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Keywords: | Amino acid biosynthesis Aspartate kinase Lysine Mutant (threonine overproduction) Threonine Zea(amino acids) |
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