The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase.
N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated 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. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.
Zapata
G
G
Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and Research, Bethesda, MD 20892.
Roller
P P
PP
Crowley
J
J
Vann
W F
WF
D001483:Base Sequence; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000216:N-Acylneuraminate Cytidylyltransferase / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D013438:Sulfhydryl Compounds / Q000378:metabolism
D017931:DNA Primers; D013438:Sulfhydryl Compounds; D000216:N-Acylneuraminate Cytidylyltransferase; D003545:Cysteine