ENHANCEMENT OF CATALYTIC ACTIVITY BY GENE TRUNCATION - ACTIVATION OF L-ASPARTASE FROM ESCHERICHIA-COLI

Aspartase front Escherichia coli is activated by proteolysis at the ca rboxy-terminal. A systematic study has been undertaken with the goal o f identifying the amino acids in this region that influence the cataly tic activity of aspartase. Stop codons have been introduced at various positions to prematurely truncate the aspA gene that encodes for aspa rtase by sequentially eliminating each. of the polar and charged amino acids in this region. The affinity of the enzyme for its substrate as partic acid decreases systematically as each functionally significant amino acid is eliminated. However, enhanced catalytic activity (up to 2.5 times the k(cat) for native aspartase) is observed for those trunc ation mutants that end in a positively charged carboxyterminal amino a cid. The precise position of the proteolytic activation of aspartase h as been defined, and this covalent activation has been shown to be ind ependent of the allosteric activation of aspartase that is also observ ed. (C) 1997 Academic Press.