title: Mechanism of phosphoglycolate phosphatase. Studies of hydrolysis and transphosphorylation, substrate analogs, and sulfhydryl inhibition.
abstract: Enzymatic hydrolysis of phosphoglycolate proceeds through O-P bond cleavage as determined by reaction in H218O and analysis of the trimethylsilyl derivatives of the reaction products by mass spectrometry. No phosphate, hydroxyl, or carboxyl exchange occurred. End product inhibition was consistent with an ordered release of products, first the alcoholic product, glycolate, then phosphate. Analysis of the data indicated that the phosphate.enzyme complex dissociated very rapidly, and this was confirmed by use of alternative phosphomonoester substrates. Maximum velocity with these alternate substrates was found to be proportional to the pKa of of the corresponding alcoholic product, indicating the rate-limiting step in the reaction was protonation of the bridge oxygen. The use of substrate analogs further suggested that enzymatic specificity residues in exacting steric requirements for binding, and that large alkyl groups were excluded on this basis. Phosphoglycolate phosphatase catalyzed transphorylation to a wide range of acceptors and was inhibited at the active site by diisopropyl-fluorophosphate. The data suggest that the reaction sequence proceeds via a phosphoenzyme intermediate. N-Ethylmaleimide slowly inactivated the enzyme, the rate being greatly increased by P-glycolate, but not by magnesium or phosphate ions. The data suggest a conformational change is necessary to induce the transition state complex and phosphoenzyme formation. This may account for the phosphate acceptor specificity and is consistent with the failure to observe an enzyme-mediated H2O-phosphate oxygen exchange.
mesh_terms: D006016:Glycolates; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010944:Plants / Q000201:enzymology; D010947:Plants, Toxic; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology
chemical_list: D006016:Glycolates; D010744:Phosphoric Monoester Hydrolases