TY - JOUR
T1 - Characterization of mutant holocarboxylase synthetase (HCS)
T2 - A K(m) for biotin was not elevated in a patient with HCS deficiency
AU - Aoki, Yoko
AU - Suzuki, Yoichi
AU - Li, Xue
AU - Sakamoto, Osamu
AU - Chikaoka, Hiroshi
AU - Takita, Seiji
AU - Narisawa, Kuniaki
N1 - Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 1997/12
Y1 - 1997/12
N2 - Holocarboxylase synthetase (HCS) is an essential enzyme for the biotinylation of several mammalian carboxylases. A deficiency of HCS is accountable for early onset biotin-responsive multiple carboxylase deficiency. To address the mechanism of biotin responsiveness, we analyzed the kinetic properties of the previously identified mutant, L237P, and another mutant, V550M, described in this report. The V550M mutant contains a G to A transition at position 1935, which is within the putative biotin binding site, whereas the mutation in L237P occurs outside the biotin binding site. K(m) and V(max) values for the mutant proteins were determined by overexpressing cDNAs encoding the mutants in transformed fibroblasts from an HCS-deficient patient. Enzyme activity assays were performed using apocarboxyl carrier protein as a substrate. A K(m) for biotin that was larger than the value found for the wild-type cDNA was observed in fibroblasts transfected with the V550M cDNA, but not the L237P cDNA. The V(max) for the expressed L237P cDNA was 4.3% of that observed for the wild-type cDNA. Biotin-responsiveness in the patient with the L237P mutation was neither due to an increased affinity for biotin nor a restoration of stability of the mutant by biotin treatment. A new mechanism of biotin responsiveness in HCS deficiency is presented.
AB - Holocarboxylase synthetase (HCS) is an essential enzyme for the biotinylation of several mammalian carboxylases. A deficiency of HCS is accountable for early onset biotin-responsive multiple carboxylase deficiency. To address the mechanism of biotin responsiveness, we analyzed the kinetic properties of the previously identified mutant, L237P, and another mutant, V550M, described in this report. The V550M mutant contains a G to A transition at position 1935, which is within the putative biotin binding site, whereas the mutation in L237P occurs outside the biotin binding site. K(m) and V(max) values for the mutant proteins were determined by overexpressing cDNAs encoding the mutants in transformed fibroblasts from an HCS-deficient patient. Enzyme activity assays were performed using apocarboxyl carrier protein as a substrate. A K(m) for biotin that was larger than the value found for the wild-type cDNA was observed in fibroblasts transfected with the V550M cDNA, but not the L237P cDNA. The V(max) for the expressed L237P cDNA was 4.3% of that observed for the wild-type cDNA. Biotin-responsiveness in the patient with the L237P mutation was neither due to an increased affinity for biotin nor a restoration of stability of the mutant by biotin treatment. A new mechanism of biotin responsiveness in HCS deficiency is presented.
UR - http://www.scopus.com/inward/record.url?scp=0030702915&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0030702915&partnerID=8YFLogxK
U2 - 10.1203/00006450-199712000-00021
DO - 10.1203/00006450-199712000-00021
M3 - Article
C2 - 9396568
AN - SCOPUS:0030702915
VL - 42
SP - 849
EP - 854
JO - Pediatric Research
JF - Pediatric Research
SN - 0031-3998
IS - 6
ER -