EFFECTS OF 2 MUTATIONS DETECTED IN MEDIUM-CHAIN ACYL-COA DEHYDROGENASE (MCAD)-DEFICIENT PATIENTS ON FOLDING, OLIGOMER ASSEMBLY, AND STABILITY OF MCAD ENZYME
P. Bross et al., EFFECTS OF 2 MUTATIONS DETECTED IN MEDIUM-CHAIN ACYL-COA DEHYDROGENASE (MCAD)-DEFICIENT PATIENTS ON FOLDING, OLIGOMER ASSEMBLY, AND STABILITY OF MCAD ENZYME, The Journal of biological chemistry, 270(17), 1995, pp. 10284-10290
We have used expression of human medium chain acyl-CoA dehydrogenase (
MCAD) in Escherichia coli as a model system for dissecting the molecul
ar effects of two mutations detected in patients with MCAD deficiency,
We demonstrate that the R28C mutation predominantly affects polypepti
de folding. The amounts of active R28C mutant enzyme produced could be
modulated between undetectable to 100% of the wild-type control by ma
nipulating the level of available chaperonins and the growth temperatu
re, For the prevalent K304E mutation, however, the amounts of active m
utant enzyme could be modulated only in a range from undetectable to a
pproximately 50% of the wild-type, and the as sembled mutant enzyme di
splayed a decreased thermal stability. Two artificially constructed mu
tants (R304Q and K304E/D346K) yielded clearly higher amounts of active
MCAD enzyme than the K304E mutant but were also responsive to chapero
nin co-overexpression and growth at low temperature. The thermal stabi
lity profile of the H304E/D346K double mutant was shifted to even lowe
r temperatures than that of the K304E mutant, whereas that of the K804
Q mutant was closely similar to the wild-type, Taken together, the res
ults show that the K304E mutation affects (i) polypeptide folding due
to elimination of the positively charged lysine and (ii) oligomer asse
mbly and stability due to replacement of lysine 304 with the negativel
y charged glutamic acid.