PMID: 7626232Apr 1, 1995

Affinity labelling of the catalytic and allosteric ATP binding sites on pyruvate kinase type I from Escherichia coli

Biological Chemistry Hoppe-Seyler
G ValentiniM L Speranza

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.

References

Citations

Oct 15, 1979·Journal of Molecular Biology·D I StuartD K Stammers
Feb 15, 1987·Archives of Biochemistry and Biophysics·G BezaresS Bazaes
Jan 1, 1983·Annual Review of Biochemistry·R F Colman
Jan 1, 1982·Methods in Enzymology·M Malcovati, G Valentini
Jan 1, 1993·Progress in Biophysics and Molecular Biology·L A Fothergill-Gilmore, P A Michels

Related Concepts

Alkalescens-Dispar Group
Allosteric Site
Borohydrides
Pyruvate Kinase
Sodium borohydride, 3H-labeled
Sequence Analysis
Enzyme Activity
Pyruvate Kinase Measurement
Blood Enzyme Activity (Lab Test)
Purification Aspects

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