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Investigation of putative active site lysine residues in hydroxymethylbilane synthase preparation and characterization of mutants in which a lysine 55 b lysine 59 and c both lysine 55 and lysine 59 have been replaced by glutamine



Investigation of putative active site lysine residues in hydroxymethylbilane synthase preparation and characterization of mutants in which a lysine 55 b lysine 59 and c both lysine 55 and lysine 59 have been replaced by glutamine



Biochemical Journal 271(2): 487-492



A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx. 1000-fold overexpression 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 kcat.app./Kmapp. 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.

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