(S)-Mandelate dehydrogenase from Pseudomonas putida: mutations of the catalytic base histidine-274 and chemical rescue of activity.
(S)-Mandelate dehydrogenase from Pseudomonas putida, an FMN-dependent alpha-hydroxy acid dehydrogenase, oxidizes (S)-mandelate to benzoylformate. The generally accepted catalytic mechanism for this enzyme involves the formation of a carbanion intermediate. Histidine-274 has been proposed to be the active-site base that abstracts the substrate alpha-proton to generate the carbanion. Histidine-274 was altered to glycine, alanine, and asparagine. All three mutants were completely inactive. The mutants were able to form adducts with sulfite, though with much weaker affinity than the wild-type enzyme. Binding of the inhibitor, (R)-mandelate, was not greatly affected by the mutation, unlike that of the substrate, (S)-mandelate, indicating that H274 plays a role in substrate binding. The activity of H274G and, to a lesser extent, H274A could be partially restored by the addition of exogenous imidazoles. The maximum rescued activity for H274G with imidazole was approximately 0.1% of the wild-type value. Saturation kinetics obtained for rescued activity suggest that formation of a ternary complex of imidazole, enzyme, and substrate is required for catalysis. pH-dependence studies confirm that the free base form of imidazole is the rescue agent. An earlier study of pH profiles of the wild-type enzyme indicated that deprotonation of a residue with a pK(a) of 5.5 in the free enzyme was essential for activity (Lehoux, I. E., and Mitra, B. (1999) Biochemistry 38, 5836-5848). Data obtained in this work confirm that the pK(a) of 5.5 belongs to histidine-274.
Lehoux
I E
IE
Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, Michigan 48201, USA.
Mitra
B
B
D000409:Alanine / Q000235:genetics; D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001216:Asparagine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D003903:Deuterium; D004791:Enzyme Inhibitors / Q000737:chemistry; D005486:Flavin Mononucleotide / Q000737:chemistry; D005998:Glycine / Q000235:genetics; D006639:Histidine / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D008333:Mandelic Acids / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013447:Sulfites / Q000737:chemistry
D004791:Enzyme Inhibitors; D007093:Imidazoles; D008333:Mandelic Acids; D011994:Recombinant Proteins; D013447:Sulfites; D006639:Histidine; D001216:Asparagine; D005486:Flavin Mononucleotide; D003903:Deuterium; D000429:Alcohol Oxidoreductases; C021538:L-mandelate dehydrogenase; C037938:mandelic acid; D000409:Alanine; D005998:Glycine