Identification of the catalytic residues involved in the carboxyl transfer of pyruvate carboxylase.
To clarify the mechanism of carboxyl transfer from carboxylbiotin to pyruvate, the following conserved amino acid residues present in the carboxyl transferase domain of Bacillus thermodenitrificans pyruvate carboxylase were converted to homologous amino acids: Asp543, Glu576, Glu592, Asp649, Lys712, Asp713, and Asp762. The carboxylase activity of the resulting mutants, D543E, E576D, E576Q, E592Q, D649N, K712R, K712Q, D713E, D713N, D762E, and D762N, was generally less than that of the wild type from mutation, but it decreased the most to 5% or even less than that of the wild type with D543E, D576Q, D649N, K712R, and K712Q. The decrease in activity observed for Asp543, Asp649, and Lys712 mutants was not for structural reasons because their structures seemed to remain intact as assessed by gel filtration and circular dichroism. On the basis of these data, a mechanism is proposed where Lys712 and Asp543 serve as the key acid and base catalyst, respectively.
Yong-Biao
Jin
J
Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Iizuka 820-8502, Japan.
Islam
Md Nurul
MN
Sueda
Shinji
S
Kondo
Hiroki
H
D000103:Acetyl-CoA Carboxylase / Q000737:chemistry*; D001224:Aspartic Acid / Q000235:genetics; D001407:Bacillus / Q000201:enzymology*; D002262:Carboxy-Lyases / Q000737:chemistry; D019878:Carboxyl and Carbamoyl Transferases / Q000737:chemistry / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry*; D020134:Catalytic Domain / Q000235:genetics; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D017124:Conserved Sequence; D004789:Enzyme Activation / Q000235:genetics; D054889:Fatty Acid Synthase, Type II; D018698:Glutamic Acid / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010072:Oxamic Acid / Q000737:chemistry; D017434:Protein Structure, Tertiary / Q000235:genetics; D011766:Pyruvate Carboxylase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D019289:Pyruvic Acid / Q000737:chemistry; D014316:Tritium / Q000737:chemistry
D002352:Carrier Proteins; D014316:Tritium; D001224:Aspartic Acid; D018698:Glutamic Acid; D019289:Pyruvic Acid; D019878:Carboxyl and Carbamoyl Transferases; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase; D054889:Fatty Acid Synthase, Type II; D011766:Pyruvate Carboxylase; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein; D008239:Lysine; D010072:Oxamic Acid