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)
Wyss
M
M
Swiss Federal Institute of Technology, Institute for Cell Biology, Zürich, Switzerland.
James
P
P
Schlegel
J
J
Wallimann
T
T
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