Functional and molecular modelling studies of two hereditary fructose intolerance-causing mutations at arginine 303 in human liver aldolase.
We have identified a novel hereditary fructose intolerance mutation in the aldolase B gene (i.e. liver aldolase) that causes an arginine-to-glutamine substitution at residue 303 (Arg(303)-->Gln). We previously described another mutation (Arg(303)-->Trp) at the same residue. We have expressed the wild-type protein and the two mutated proteins and characterized their kinetic properties. The catalytic efficiency of protein Gln(303) is approx. 1/100 that of the wild-type for substrates fructose 1,6-bisphosphate and fructose 1-phosphate. The Trp(303) enzyme has a catalytic efficiency approx. 1/4800 that of the wild-type for fructose 1,6-bisphosphate; no activity was detected with fructose 1-phosphate. The mutation Arg(303)-->Trp thus substitution impairs enzyme activity more than Arg(303)-->Gln. Three-dimensional models of wild-type, Trp(303) and Gln(303) aldolase B generated by homology-modelling techniques suggest that, because of its larger size, tryptophan exerts a greater deranging effect than glutamine on the enzyme's three-dimensional structure. Our results show that the Arg(303)-->Gln substitution is a novel mutation causing hereditary fructose intolerance and provide a functional demonstration that Arg(303), a conserved residue in all vertebrate aldolases, has a dominant role in substrate binding during enzyme catalysis.
Santamaria
R
R
Dipartimento di Biochimica e Biotecnologie Mediche, CEINGE-Biotecnologie Avanzate, Università di Napoli 'Federico II', Via S. Pansini 5, I-80131, Napoli, Italy.
Esposito
G
G
Vitagliano
L
L
Race
V
V
Paglionico
I
I
Zancan
L
L
Zagari
A
A
Salvatore
F
F
D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis; D017931:DNA Primers; D005260:Female; D005633:Fructose Intolerance / Q000235:genetics*; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007223:Infant; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008958:Models, Molecular; D009154:Mutation; D010375:Pedigree; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D011994:Recombinant Proteins; D001120:Arginine; D005634:Fructose-Bisphosphate Aldolase