A misfolded but active dimer of bovine seminal ribonuclease.
Bovine seminal ribonuclease (BS-RNase) is an unusual homolog of RNase A. Isolated from bulls as a dimer, BS-RNase has special biological properties including antispermatogenic, antitumor and immunosuppressive activities. The structural bases for these properties are unknown. Four forms of BS-RNase were isolated after folding and air oxidation of the denatured and reduced protein produced in Escherichia coli: two dimers (M = M and M x I, where x signifies an active site composed of residues from both subunits) and two monomers (M and I). Considerable ribonuclease activity was generated by air oxidation of an equimolar mixture of two inactive mutant proteins ([H12D]BS-RNase and [H119D]BS-RNase) prepared by site-directed mutagenesis. This activity came from a dimer (M x I) with a composite active site. 1H-NMR spectroscopy revealed that this dimer contained one correctly folded subunit (M), and one incorrectly folded subunit (I). Form I, which is a poor catalyst, was activated by ribonuclease S-protein, suggesting that the C-terminal portion of I was not folded properly. Electrospray-ionization mass spectrometry and sulfhydryl group titration indicated that I contains a single oxidized sulfhydryl group, which cannot participate in a disulfide bond. These results show that quaternary structure in BS-RNase is attained by the initial formation of two monomers, M and I, which then combine with another M to form M = M and M x I, respectively. Adventitious oxidation can thus lead to the formation of a misfolded but active enzyme (M x I).
Kim
J S
JS
Department of Biochemistry, University of Wisconsin-Madison 53706-1569.
Raines
R T
RT
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000494:pharmacology; D011108:Polymers; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012259:Ribonuclease, Pancreatic / Q000494:pharmacology; D012661:Semen / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000378:metabolism
D010446:Peptide Fragments; D011108:Polymers; D013438:Sulfhydryl Compounds; C060871:ribonuclease A S-peptide; D004722:Endoribonucleases; C032523:ribonuclease SPL; D012259:Ribonuclease, Pancreatic