Two novel variants of human medium chain acyl-CoA dehydrogenase (MCAD). K364R, a folding mutation, and R256T, a catalytic-site mutation resulting in a well-folded but totally inactive protein.
Two novel rare mutations, MCAD approximately 842G-->C (R256T) and MCAD approximately 1166A-->G (K364R), have been investigated to assess how far the biochemical properties of the mutant proteins correlate with the clinical phenotype of medium chain acyl-CoA dehydrogenase (MCAD) deficiency. When the gene for K364R was overexpressed in Escherichia coli, the synthesized mutant protein only exhibited activity when the gene for chaperonin GroELS was co-overexpressed. Levels of activity correlated with the amounts of native MCAD protein visible in western blots. The R256T mutant, by contrast, displayed no activity either with or without chaperonin, but in this case a strong MCAD protein band was seen in the western blots throughout. The proteins were also purified, and the enzyme function and thermostability investigated. The K364R protein showed only moderate kinetic impairment, whereas the R256T protein was again totally inactive. Neither mutant showed marked depletion of FAD. The pure K364R protein was considerably less thermostable than wild-type MCAD. Western blots indicated that, although the R256T mutant protein is less thermostable than normal MCAD, it is much more stable than K364R. Though clinically asymptomatic thus far, both mutations have a severe impact on the biochemical phenotype of the protein. K364R, like several previously described MCAD mutant proteins, appears to be defective in folding. R256T, by contrast, is a well-folded protein that is nevertheless devoid of catalytic activity. How the mutations specifically affect the catalytic activity and the folding is further discussed.
O'Reilly
Linda P
LP
Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland.
Andresen
Brage S
BS
Engel
Paul C
PC
D042964:Acyl-CoA Dehydrogenase / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D019943:Amino Acid Substitution; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D018833:Chaperonins / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D010641:Phenotype; D017354:Point Mutation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry
D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D004247:DNA; D042964:Acyl-CoA Dehydrogenase; D018833:Chaperonins