title: Affinity labelling of the catalytic and allosteric ATP binding sites on pyruvate kinase type I from Escherichia coli.
abstract: The allosterically regulated pyruvate kinase type I (PKI) from E. coli was inactivated by the ATP analog 2',3'-dialdehyde ATP (o-ATP) with a Ki of 3.6 mM. ATP and phosphoenolpyruvate protected the enzyme activity while the allosteric activator fructose 1,6-bisphosphate enhanced the rate of inactivation. Incubation with o-ATP, followed by reduction of the formed Schiff bases with radioactive sodium borohydride, was employed to determine the ATP binding sites of PKI. After tryptic digestion, the purification of the labelled peptides and the sequence analysis allowed to identify four modified lysyl residues, namely Lys173, Lys175, Lys272, and Lys317 of the known DNA-deduced sequence of PKI. The close lysines 173 and 175 reacted with o-ATP in a mutually exclusive way and accounted together for 53% of the recovered radioactivity, the rest being distributed on Lys272 (31%) and Lys317 (16%). When fitted on the available three-dimensional structure of muscle pyruvate kinase, the position of the modified lysines defines both the catalytic and the allosteric ATP binding sites on PKI.
authors.affiliation: Dipartimento di Biochimica, Università di Pavia, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Valentini
authors.forename: P
authors.initials: P
authors.lastname: Iadarola
authors.forename: G
authors.initials: G
authors.lastname: Ferri
authors.forename: M L
authors.initials: ML
authors.lastname: Speranza
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels; D000495:Allosteric Site; D000595:Amino Acid Sequence; D001894:Borohydrides; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011770:Pyruvate Kinase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D001894:Borohydrides; C017199:2',3'-dialdehyde ATP; C025364:sodium borohydride; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase