Limited proteolysis of creatine kinase. Implications for three-dimensional structure and for conformational substrates.
Proteinase K, subtilisin, pronase E, elastase, bactotrypsin, and thermolysin are all shown here to cleave native mitochondrial creatine kinase from chicken heart (Mib-CK) very specifically at a single site, either before or after Ala-323. In analogy with hen egg ovalbumin, where the same proteases all cleaved the polypeptide chain very specifically around Ala-352, Ala-323 of Mib-CK may be located in an exposed surface loop that is sensitive to protease attack. Gel permeation chromatography demonstrated that the two proteolytic fragments of Mib-CK with M(r)'s of approximately 37,000 and approximately 6000 remain associated with each other. Proteinase K cleavage did not influence the octamer to dimer ratio of Mib-CK, indicating that selective cleavage after Ala-323 has no direct effect on dimer-dimer interfaces within the octamer. However, upon addition of MgADP plus creatine and nitrate to induce a transition-state analogue complex of the enzyme, native Mib-CK dissociated much more readily into dimers than proteinase K-digested Mib-CK. Furthermore, proteinase K cleavage of Mib-CK resulted in 2-11-fold decreases in the Vmax values, as well as in 6-23-fold increases in the Km values for phosphocreatine, creatine, and MgATP, whereas the Kd values for both MgATP and creatine were unaffected. Consequently, proteinase K cleavage of Mib-CK does not affect substrate binding per se, but interferes with substrate-induced conformational changes which are essential for catalysis and which mediate the synergism in substrate binding as it is observed with the unmodified enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Swiss Federal Institute of Technology, Institute for Cell Biology, Zürich, Switzerland.
M
M
Wyss
P
P
James
J
J
Schlegel
T
T
Wallimann
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002850:Chromatography, Gel; D003402:Creatine Kinase / Q000737:chemistry*; D010450:Endopeptidases / Q000378:metabolism*; D005260:Female; D007527:Isoenzymes; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010735:Phosphoglycerate Kinase / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity
D007527:Isoenzymes; D010446:Peptide Fragments; D010047:Ovalbumin; D010735:Phosphoglycerate Kinase; D003402:Creatine Kinase; D010450:Endopeptidases