Site-directed alteration of the active-site residues of histidine decarboxylase from Clostridium perfringens.
To clarify the mechanism of biogenesis and catalysis by the pyruvoyl-dependent histidine decarboxylase (HisDCase) from Clostridium perfringens, 12 mutant genes encoding amino acid substitutions at the active site of this enzyme were constructed and expressed in Escherichia coli. The resulting mutant proteins were purified to homogeneity, characterized, and subjected to kinetic analysis. The results (a) exclude all polar amino acid residues in the active site except Glu-214 as donor of the proton that replaces the carboxyl group of histidine during decarboxylation and, since E214I and E214H are nearly inactive, indicate that Glu-214 is the essential proton donor; (b) demonstrate the importance to substrate binding of hydrophobic interactions between Phe-98, Ile-74, and the imidazole ring of histidine, and of hydrogen bonding between Asp-78 and N2 of the substrate; and (c) demonstrate a significant unidentified role for Glu-81 in the maintenance of the active-site structure. The proposed roles of these amino acid residues are consistent with those assigned on the basis of crystallographic evidence to the corresponding residues at the active site of the related HisDCase from Lactobacillus 30a [Gallagher, T., Snell, E. E., & Hackert, M. L. (1989) J. Biol. Chem. 264, 12737-12743]. Of the residues altered, only Ser-97 was essential for the autocatalytic serinolysis reaction by which this HisDCase, (alpha beta)6, is derived from its inactive, pyruvate-free precursor, proHisDCase, pi 6.(ABSTRACT TRUNCATED AT 250 WORDS)
van Poelje
P D
PD
Department of Microbiology, University of Texas, Austin 78712.
Kamath
A V
AV
Snell
E E
EE
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003016:Clostridium perfringens / Q000201:enzymology / Q000235:genetics*; D004789:Enzyme Activation; D006640:Histidine Decarboxylase / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D006640:Histidine Decarboxylase