Site-directed mutagenesis of the alpha subunit of human prolyl 4-hydroxylase. Identification of three histidine residues critical for catalytic activity.
Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer in which the alpha subunits contribute to most parts of the two catalytic sites. To study the roles of histidine and cysteine residues in this catalytic activity we converted all 5 histidines that are conserved between species, 4 nonconserved histidines, and 3 conserved cysteines of the human alpha subunit individually to serine and expressed the mutant alpha subunits together with the wild-type beta subunit in insect cells by means of baculovirus vectors. Mutation of any of the 3 conserved histidines, residues 412, 483, and 501, inactivated the enzyme completely or essentially completely, with no effect on tetramer assembly or binding of the tetramer to poly(L-proline). These histidines are likely to provide the three ligands needed for the binding of Fe2+ to a catalytic site. Mutation of either of the other 2 conserved histidines reduced the amount of enzyme tetramer by 20-25% and the activity of the tetramer by 30-60%. Mutation of the nonconserved histidine 324 totally prevented tetramer assembly, whereas mutation of the 3 other nonconserved histidines had no effects. Two of the 3 cysteine to serine mutations, those involving residues 486 and 511, totally prevented tetramer assembly under the present conditions, whereas the third, involving residue 150, had only a minor effect in reducing tetramer assembly and activity. The data do not support previous suggestions that cysteine residues are involved in Fe2+ binding sites. Additional mutagenesis experiments demonstrated that the two glycosylated asparagines have no role in tetramer assembly or catalytic activity.
Lamberg
A
A
Collagen Research Unit, University of Oulu, Finland.
Pihlajaniemi
T
T
Kivirikko
K I
KI
D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D005973:Glutamine; D012694:Serine; D006639:Histidine; D001216:Asparagine; D011393:Procollagen-Proline Dioxygenase