Prolyl 4-hydroxylase: defective assembly of alpha-subunit mutants indicates that assembled alpha-subunits are intramolecularly disulfide bonded.
The vital hydroxylation of peptidyl proline residues in collagens and protein with collagen-like amino acid sequences is catalyzed by the tetrameric enzyme prolyl 4-hydroxylase (P4-H). We have previously detailed [John et al. (1993) EMBO J. 12, 1587-1595] the redox-dependent assembly of the catalytically important alpha-subunit (64 kDa) in a cell-free system containing endogenous beta-subunits (PDI, 60 kDa). To identify the origin of this redox-dependent assembly, we have now shown directly by an electrophoretic mobility shift assay that the assembled wild-type protein possesses at least one intramolecular disulfide bond. We also analyzed five alpha-subunit mutants that have single Cys to Ser mutations in one of the five Cys residues present in the wild-type protein and found that (i) subunits mutated at Cys150 or Cys511 formed intramolecular disulfide bonds, whereas subunits mutated at Cys276, Cys293, or Cys486 did not, (ii) mutation of Cys276, Cys293, or Cys486 led to a large reduction in alpha-beta complex formation, (iii) subunits mutated at Cys276, Cys293, Cys486, or Cys511 were recognized by an antiserum raised against an alpha-subunit C-terminal peptide which failed to recognize the assembled wild-type subunit or the assembled subunit mutated at Cys150, and (iv) the assembled complexes fractionated in a similar position to the purified protein on sucrose gradients whereas the assembly-defective mutants formed higher molecular weight aggregates or complexes with other proteins. On the basis of these results, we propose that P4-H alpha-subunits possess an intramolecular disulfide bond between Cys276 and Cys293 that is essential for alpha-beta complex formation.
John
D C
DC
School of Biological Sciences, University of Manchester, U.K.
Bulleid
N J
NJ
D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry* / Q000235:genetics; D014176:Protein Biosynthesis; D011817:Rabbits; D051381:Rats; D012694:Serine / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
D004220:Disulfides; D046911:Macromolecular Substances; D012694:Serine; D011393:Procollagen-Proline Dioxygenase; D003545:Cysteine