The organ common-type (CT) isoenzyme of acylphosphatase is inactivated by Woodward's reagent K (WRK) (N-ethyl-5-phenylisoxazolium-3ʹ-sulphonate) at pH 6.0. The inactivation reaction follows apparent pseudo first-order kinetics. The dependence of the reciprocal of the pseudo first-order kinetic constant (kobs) on the reciprocal WRK concentration reveals saturation kinetics, suggesting that the WRK forms a reversible complex with the enzyme before causing inactivation. Competitive inhibitors, such as inorganic phosphate and ATP, protect the enzyme from WRK inactivation, suggesting that this reagent acts at or near to the enzyme active site. The reagent-enzyme adduct, which elicits a strong absorption band with λmax at 346 nm, was separated from unreacted enzyme by reverse phase HPLC and the modified protein was cleaved with endoproteinase Glu-C to produce fragments. The HPLC fractionation gave two reagent-labelled peptides (peak 1 and peak 2) that were analysed by ion-spray MS and sequenced. The former is VFFRKHTQAE (residues 20-29 of human CT acylphosphatase) and the latter IFGKVQGVFFRKHTQAE (residues 13-29). MS demonstrated that both peptides are WRK adducts. A fragment ion with m/z of 1171, which is present in the mass spectrum of peak 1, has been identified as a WRK adduct of the peptide fragment 20-26. The λmax at 346 nm of WRK adduct suggests that the modified residue is His-25. Five recombinant enzymes mutated in residues included in the 20-29 polypeptide stretch have been produced. Analysis of their reactivities with WRK demonstrates that His-25 is the molecular target of the reagent as its modification causes the inactivation of the enzyme. Since both His-25 → Gln and His-25 → Phe mutants maintain high catalytic activity, we suggest that the observed enzyme inactivation is caused by the reagent (covalently bound to His-25), which shields the active site.
Mechanism of acylphosphatase inactivation by Woodward's reagent K
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Paolo PAOLI, Tania FIASCHI, Paolo CIRRI, Guido CAMICI, Giampaolo MANAO, Gianni CAPPUGI, Giovanni RAUGEI, Gloriano MONETI, Giampietro RAMPONI; Mechanism of acylphosphatase inactivation by Woodward's reagent K. Biochem J 15 December 1997; 328 (3): 855–861. doi: https://doi.org/10.1042/bj3280855
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