Characterization of four variant forms of human propionyl-CoA carboxylase expressed in Escherichia coli.
Propionyl-CoA carboxylase (PCC) is a biotin-dependent mitochondrial enzyme that catalyzes the conversion of propionyl-CoA to D-methylmalonyl-CoA. PCC consists of two heterologous subunits, alpha PCC and beta PCC, which are encoded by the nuclear PCCA and PCCB genes, respectively. Deficiency of PCC results in a metabolic disorder, propionic acidemia, which is sufficiently severe to cause neonatal death. We have purified three PCCs containing pathogenic mutations in the beta subunit (R165W, E168K, and R410W) and one PCCB polymorphism (A497V) to homogeneity to elucidate the potential structural and functional effects of these substitutions. We observed no significant difference in Km values for propionyl-CoA between wild-type and the variant enzymes, which indicated that these substitutions had no effect on the affinity of the enzyme for this substrate. Furthermore, the kinetic studies indicated that mutation R410W was not involved in propionyl-CoA binding in contrast to a previous report. The three mutant PCCs had half the catalytic efficiency of wild-type PCC as judged by the kcat/Km ratios. No significant differences have been observed in molecular mass or secondary structure among these enzymes. However, the variant PCCs were less thermostable than the wild-type. Following incubation at 47 degrees C, blue native-PAGE revealed a lower oligomeric form (alpha2beta2) in the three mutants not detectable in wild-type and the polymorphism. Interestingly, the lower oligomeric form was also observed in the corresponding crude Escherichia coli extracts. Our biochemical data and the structural analysis using a beta PCC homology model indicate that the pathogenic nature of these mutations is more likely to be due to a lack of assembly rather than disruption of catalysis. The strong favorable effect of the co-expressed chaperone proteins on PCC folding, assembly, and activity suggest that propionic acidemia may be amenable to chaperone therapy.
Jiang
Hua
H
Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.
Rao
K Sudhindra
KS
Yee
Vivien C
VC
Kraus
Jan P
JP
D000214:Acyl Coenzyme A / Q000737:chemistry; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D002384:Catalysis; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005838:Genotype; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D043586:Methylmalonyl-CoA Decarboxylase / Q000096:biosynthesis* / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D010641:Phenotype; D011110:Polymorphism, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014466:Ultraviolet Rays
D000214:Acyl Coenzyme A; D011994:Recombinant Proteins; C015357:methylmalonyl-coenzyme A; C009061:propionyl-coenzyme A; D001710:Biotin; D043586:Methylmalonyl-CoA Decarboxylase