Affinity labeling of the active site of yeast pyruvate kinase by 5'-p-fluorosulfonylbenzoyl adenosine.

摘要: Abstract Yeast pyruvate kinase is irreversibly inactivated by 1.1 mM 5'-p-fluorosulfonylbenzoyl adenosine at pH 8.6 with an initial rate constant of 0.019 min-1. A plot kinact versus the concentration yields a hyperbolic curve indicative binding analog prior to reaction. Marked protection afforded phosphoenolpyruvate + fructose 1,6-diphosphate Mg2+ or MgATP suggesting that reaction occurs within active site. When assayed less than saturating concentrations, inactivation caused reagent in absence added ligands appears slower, and presence phosphoenolpyruvate, 1,6-diphosphate, produces activation enzyme, extent which dependent on assay phosphoenolpyruvate. The for was observed be 0.113 activated enzyme exhibits both lowered K0.5 Hill coefficient compared native kinase. Subsequent addition leads independent Covalent thus two distinct sites. In Mg2+, incorporation tritiated linearly proportional 4 mol bound/mol tetrameric maximally enzyme. ligands, approximately 4.5 are incorporated/mol 15 min reaction, while 80% original activity remains. 8 bound 100% activaton. 1,6-diphospate, 3 tyrosines 1 lysine residue, 6 2 residues modified, amino acids nucleotide