Identification of Arg-12 in the active site of Escherichia coli K1CMP-sialic acid synthetase

Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of C MP-sialic acid from CTP and sialic acid. The active site of the 418 amino a cid E, coli enzyme was localized to its N-terminal half. The bacterial CMP- sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, a t their N-termini. Several basic residues have been identified at or near t he active site of the E. coli enzyme by chemical modification and site-dire cted mutagenesis. Only one of the lysines in the N-terminal motif, Lys-21, appears to be essential for activity. Mutation of Lys-21 in the N-terminal motif results in an inactive enzyme. Furthermore, Arg-12 of the N-terminal motif appears to be an active-site residue, based on the following evidence . Substituting Arg-12 with glycine or alanine resulted in inactive enzymes, indicating that this residue is required for enzymic activity. The Arg-12 --> Lys mutant was partially active, demonstrating that a positive charge i s required at this site. Steady-state kinetic analysis reveals changes in K -cat, K-m and K-s for CTP, which implicates Arg-12 in catalysis and substra te binding.