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J. Biol. Chem., Vol. 280, Issue 30, 27719-27727, July 29, 2005

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Characterization of Four Variant Forms of Human Propionyl-CoA Carboxylase Expressed in Escherichia coli^*

Hua Jiang, K. Sudhindra Rao, Vivien C. Yee, and Jan P. Kraus¶

From the Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado 80045 and the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106

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, PCC and 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 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 K_m 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 k_cat/K_m 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 °C, blue native-PAGE revealed a lower oligomeric form (₂₂) 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 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.

Received for publication, November 24, 2004 , and in revised form, May 12, 2005.

^* This work was supported by National Institutes of Health NICHD Grant P01HD08315 (to J. P. K.) and National Science Foundation MCB Grant 0401654 (to V. C. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Pediatrics, University of Colorado School of Medicine, Mail Stop 8313, P. O. Box 6511, Aurora, CO 80045-0511. Tel.: 303-724-3812; Fax: 303-724-3838; E-mail: jan.kraus{at}uchsc.edu.

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