The ATP/AMP binding site of pyruvate,phosphate dikinase: selective modification with fluorescein isothiocyanate.
Pyruvate,phosphate dikinase from Propionibacterium shermanii is strongly inhibited by fluorescein 5'-isothiocyanate (FITC). The time course of inactivation is biphasic, but the dependence of the pseudo-first-order rate constants on the inhibitor concentration indicates the formation of a reversible complex with the enzyme prior to covalent modification. The substrate/product nucleotide pairs MgATP and MgAMP protected against inactivation, while in the absence of Mg2+, both the nucleotides were ineffective. Previously, an essential lysine at the ATP/AMP subsite of the enzyme from Bacteroides symbiosus had been implicated by use of the 2',3'-dialdehyde of AMP (oAMP) [Evans, C. T., Goss, N. H., & Wood, H. G. (1980) Biochemistry 19, 5809]. The inhibition by FITC was competitive with MgAMP, and a multiple inhibition analysis plot indicated that binding of oAMP and FITC was mutually exclusive. These observations suggest that FITC and oAMP bind at the nucleotide binding site and probably to the same reactive lysine that is modified by oAMP. With peptide mapping by high-performance liquid chromatography, FITC was found to be a suitable probe for isolating the peptide from the ATP/AMP subsite.
Phillips
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Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology; D007700:Kinetics; D010449:Peptide Mapping; D010770:Phosphotransferases / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D011772:Pyruvate, Orthophosphate Dikinase / Q000037:antagonists & inhibitors*; D013861:Thiocyanates / Q000494:pharmacology*; D014357:Trypsin
D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D011772:Pyruvate, Orthophosphate Dikinase; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate