title: Investigation of putative active-site lysine residues in hydroxymethylbilane synthase. Preparation and characterization of mutants in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 have been replaced by glutamine.
abstract: A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx. 1000-fold over-expression of hydroxymethylbilane synthase (HMBS). This construct was used to generate HMBS in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 were replaced by glutamine (K55Q, K59Q and K55Q-K59Q respectively). All three modified enzymes are chromatographically separable from wild-type enzyme. Kinetic studies showed that the substitution K55Q has little effect whereas K59Q causes a 25-fold decrease in Kapp. cat./Kapp. m. Treatment of K55Q, K59Q and K55Q-K59Q separately with pyridoxal 5'-phosphate and NaBH4 resulted in incomplete and non-specific reaction with the remaining lysine residues. Pyridoxal modification of Lys-59 in the K55Q mutant caused greater enzymic inactivation than similar modification of Lys-55 in K59Q. The results in sum show that, though Lys-55 and Lys-59 may be at or near the active site, neither is indispensable for the catalytic activity of HMBS.
authors.affiliation: University of Cambridge Chemical Laboratory, U.K.
authors.forename: A
authors.initials: A
authors.lastname: Hädener
authors.forename: P R
authors.initials: PR
authors.lastname: Alefounder
authors.forename: G J
authors.initials: GJ
authors.lastname: Hart
authors.forename: C
authors.initials: C
authors.lastname: Abell
authors.forename: A R
authors.initials: AR
authors.lastname: Battersby
mesh_terms: D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D005973:Glutamine; D011163:Hydroxymethylbilane Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D014169:Transformation, Bacterial
chemical_list: D005973:Glutamine; D011163:Hydroxymethylbilane Synthase; D008239:Lysine