Identification of Arg-12 in the active site of Escherichia coli K1 CMP-sialic acid synthetase
| Jazyk: | English |
|---|---|
| Přístupová URL adresa: | https://explore.openaire.eu/search/publication?articleId=pmid________::6f7df466b657b8a9000bf9e1a8ea9b65 https://europepmc.org/articles/PMC1220567/ |
| Rights: | OPEN |
| Přírůstkové číslo: | edsair.pmid..........6f7df466b657b8a9000bf9e1a8ea9b65 |
| Autor: | Stoughton, D M, Zapata, G, Picone, R, Vann, W F |
| Jazyk: | angličtina |
| Rok vydání: | 1999 |
| Předmět: |
N-Acylneuraminate Cytidylyltransferase
Protein Denaturation Binding Sites Cytidine Triphosphate Lysine Recombinant Fusion Proteins Amino Acid Motifs Molecular Sequence Data Arginine carbohydrates (lipids) Kinetics Amino Acid Substitution Pyridoxal Phosphate Enzyme Stability Escherichia coli Mutagenesis Site-Directed Thermodynamics Amino Acid Sequence Oxidation-Reduction Sequence Alignment Conserved Sequence Research Article |
| Popis: | Escherichia coli K1 CMP-sialic acid synthetase catalyses the synthesis of CMP-sialic acid from CTP and sialic acid. The active site of the 418 amino acid E. coli enzyme was localized to its N-terminal half. The bacterial CMP-sialic acid synthetase enzymes have a conserved motif, IAIIPARXXSKGLXXKN, at their N-termini. Several basic residues have been identified at or near the active site of the E. coli enzyme by chemical modification and site-directed 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 is 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 substrate binding. |
| Databáze: | OpenAIRE |
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