Lysine-insensitive aspartate kinase in two threonine-overproducing mutants of maize

• Published in: Planta 1990-11, Vol.182 (4), p.546-552
• Main Authors: Dotson, Stanton B., Frisch, David A., Somers, David A., Gengenbach, Burle G.
• Format: Article
• Language: English
• Subjects: Alleles; Amino acids; Barley; Corn; Enzymes; Genetic loci; Genetic mutation; Genotypes; Kinetics; Overproduction
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/BF02341030

Aspartate kinase (AK; EC 2.7.2.4) 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 and Ask2-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 homozygous Ask2 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 μM L-lysine required for 50% inhibition. In contrast, AK purified from the isogenic heterozygous Ask and homozygous Ask2 mutants required 25 and 760 μM lysine for 50% inhibition, respectively, indicating that Ask and Ask2 were separate structural loci for lysine-regulated AK subunits in maize. Further characterization of purified AK from the homozygous mutant Ask2 line indicated altered substrate and lysine inhibition kinetics. The apparent Hill coefficient was 0.7 for the mutant Ask2 AK compared with 1.6 for the wildtype enzyme, indicating that the mutant allele conferred the loss of a lysine-binding site to the mutant AK. Lysine appeared to be a linear noncompetitive inhibitor of Ask2 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 the Ask2 gene product appeared to reduce the lysine inhibition of all of the AK activity detected in homozygous Ask2 plants, indicating that maize AK is a heteromeric enzyme consisting of the two lysine-sensitive polypeptides derived from the Ask and Ask2 structural genes.