Modification of the proteolytic fragmentation pattern upon oxidation of cysteines from ribulose 1,5-bisphosphate carboxylase/oxygenase.
The proteolytic susceptibility of the native CO(2)-fixing photosynthetic enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco) has been shown to increase in vitro after oxidative treatments that affect cysteine thiols. A limited incubation of oxidized (pretreated with the disulfide cystamine) Rubisco from Chlamydomonas reinhardtii with subtilisin or proteinase K generated fragments of molecular mass about 53 kDa (band I in SDS-PAGE) and 47 kDa (band II) derived from the large subunit (55 kDa) of the enzyme. In contrast, proteolysis of the reduced Rubisco (pretreated with the free thiol cysteamine) produced only the 53 kDa band. The same fragmentation pattern was reproduced with Rubiscos from other algae and higher plants, as well as with other chemical modifications of protein cysteines. N-terminal sequencing of the fragments showed that band I arised from clipping the unstructured N-terminal stretch of the large subunit up to Lys18. Band II was generated by a cleavage close to Val69. The increased susceptibility of the oxidized form resulted from proteases gaining access to a loop (from Ser61 to Thr68) located between stretches of secondary structure that form the N-terminal domain. Native electrophoresis and kinetic analysis of fragment accumulation during subtilisin digestion demonstrated that subunit dissociation was induced by the proteolytic processing at the Ser61-Thr68 loop, which is characteristic of the oxidized Rubisco. Holoenzyme dissasembly was readily followed by the full degradation of the released subunits. In contrast, the limited processing to band I observed with the reduced enzyme did not compromise the quaternary structure of the Rubisco hexadecamer, thus preventing further proteolysis.
Marín-Navarro
Julia
J
Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de València Av. Dr. Moliner 50, Burjassot, València E-46100, Spain.
Moreno
Joaquín
J
D000595:Amino Acid Sequence; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D005056:Euglena gracilis / Q000201:enzymology; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000378:metabolism
D020035:Holoenzymes; D010446:Peptide Fragments; D021122:Protein Subunits; D010450:Endopeptidases; D012273:Ribulose-Bisphosphate Carboxylase; D003545:Cysteine