THE ROLE OF CYSTEINE RESIDUES 129 AND 329 IN ESCHERICHIA-COLI K1 CMP-NEUAC SYNTHASE

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Esc herichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catal ytic activity and structure of the protein has been investigated by si te-directed mutagenesis and chemical modification. The enzyme is inact ivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. S ite-directed mutagenesis of either residue Cys-129 to serine or Cys-32 9 to selected amino acids has minor effects on the specific activity o f the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. T he limited reactivity of the enzyme to other thiol-blocking reagents s uggests that its cysteine residues are partially exposed. The accessib ility and role of the cysteine residues in enzyme structure were inves tigated by fluorescence, c.d. and denaturation studies of wild-type an d mutant enzymes. The mutation of Cys-129 to serine makes the enzyme m ore sensitive to heat and chemical denaturation, but does not cause gr oss changes in the protein structure as judged by the c.d. spectrum. T he mutant containing Ser-129 instead of Cys-129 has a complex denatura tion pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthas e consisting of several partially denatured states. Cys-329 reacts mor e readily with N-[C-14]ethylmaleimide when the enzyme is in a heat-ind uced relaxed state. Cys-129 is less reactive and is probably a buried residue.