Abstract
Aspartate kinase is a feedback-regulated enzyme that controls the first step common to the biosynthesis of lysine, threonine, isoleucine, and methionine in plants. Aspartate kinase was purified from Black Mexican Sweet maize (Zea mays L.) cell suspension cultures for physical and kinetic characterization studies. Partial purification and elution from an anion exchange column resolved two lysine-sensitive aspartate kinase isoforms. Both isoforms were purified >1,200-fold to a minimum specific activity of 18 units/milligram of protein. Both isoforms were sensitive to the lysine analogues S-2-aminoethyl-l-cysteine, l-lysine ethyl ester, and delta-hydroxylysine. No threonine-sensitive form of aspartate kinase was detected at any stage during the purification. Additional purification steps were combined with preparative gel electrophoresis to obtain apparently homogeneous lysine-sensitive aspartate kinase. Aspartate kinase appeared to be a tetramer with a holoenzyme molecular weight of 254,000 and to be composed of 49,000 and 60,000 subunits. The tetramer appeared to disassociate during native gel electrophoresis to 113,000 dalton species that retained aspartate kinase activity.