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)

[1]  T. Wallimann,et al.  Creatine kinase: the reactive cysteine is required for synergism but is nonessential for catalysis. , 1993, Biochemistry.

[2]  M Gerstein,et al.  Domain closure in adenylate kinase. Joints on either side of two helices close like neighboring fingers. , 1993, Journal of molecular biology.

[3]  H. Eppenberger,et al.  Expression of active octameric chicken cardiac mitochondrial creatine kinase in Escherichia coli. , 1992, The Biochemical journal.

[4]  M. Wyss,et al.  Mitochondrial creatine kinase: a key enzyme of aerobic energy metabolism. , 1992, Biochimica et biophysica acta.

[5]  K Harlos,et al.  Crystal structure of the binary complex of pig muscle phosphoglycerate kinase and its substrate 3‐phospho‐D‐glycerate , 1992, Proteins.

[6]  M. Wyss,et al.  Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the 'phosphocreatine circuit' for cellular energy homeostasis. , 1992, The Biochemical journal.

[7]  G. Morris,et al.  Identification by protein microsequencing of a proteinase-V8-cleavage site in a folding intermediate of chick muscle creatine kinase. , 1991, The Biochemical journal.

[8]  H. Eppenberger,et al.  Crystallization of mitochondrial creatine kinase. Growing of large protein crystals and electron microscopic investigation of microcrystals consisting of octamers. , 1991, The Journal of biological chemistry.

[9]  G. Morris,et al.  Monoclonal antibody studies suggest a catalytic site at the interface between domains in creatine kinase. , 1990, Biochimica et biophysica acta.

[10]  R. Huber,et al.  Crystal structure of plakalbumin, a proteolytically nicked form of ovalbumin. Its relationship to the structure of cleaved alpha-1-proteinase inhibitor. , 1990, Journal of molecular biology.

[11]  R. Vale.  Microtubule-based motor proteins. , 1990, Current opinion in cell biology.

[12]  R. Carrell,et al.  Implications of the three-dimensional structure of alpha 1-antitrypsin for structure and function of serpins. , 1989, Biochemistry.

[13]  P. Stein,et al.  Ovalbumin and angiotensinogen lack serpin S-R conformational change. , 1989, The Biochemical journal.

[14]  O. Ptitsyn,et al.  Correlation between enzyme activity and hinge-bending domain displacement in 3-phosphoglycerate kinase. , 1989, European journal of biochemistry.

[15]  G. Morris.  Monoclonal antibody studies of creatine kinase. The ART epitope: evidence for an intermediate in protein folding. , 1989, The Biochemical journal.

[16]  H. Eppenberger,et al.  Purification of brain-type creatine kinase (B-CK) from several tissues of the chicken: B-CK subspecies. , 1989, Enzyme.

[17]  M. Wyss,et al.  Native mitochondrial creatine kinase forms octameric structures. I. Isolation of two interconvertible mitochondrial creatine kinase forms, dimeric and octameric mitochondrial creatine kinase: characterization, localization, and structure-function relationships. , 1988, The Journal of biological chemistry.

[18]  P. Stein,et al.  Hormone binding globulins undergo serpin conformational change in inflammation , 1988, Nature.

[19]  G. Mocz,et al.  A map of photolytic and tryptic cleavage sites on the beta heavy chain of dynein ATPase from sea urchin sperm flagella , 1988, The Journal of cell biology.

[20]  M. Wyss,et al.  Distinct tissue specific mitochondrial creatine kinases from chicken brain and striated muscle with a conserved CK framework. , 1988, Biochemical and biophysical research communications.

[21]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[22]  O. Marcillat,et al.  Only one of the two interconvertible forms of mitochondrial creatine kinase binds to heart mitoplasts. , 1987, Biochimica et biophysica acta.

[23]  C. D. dos Remedios,et al.  Structural and functional domains on actin , 1986, BioEssays : news and reviews in molecular, cellular and developmental biology.

[24]  K. Wilson,et al.  Specific proteolytic modification of creatine kinase isoenzymes. Implication of C-terminal involvement in enzymic activity but not in subunit-subunit recognition. , 1986, The Biochemical journal.

[25]  G. Morris,et al.  Monoclonal-antibody studies of creatine kinase. The proteinase K-cleavage site. , 1985, The Biochemical journal.

[26]  H. Miziorko,et al.  Differential sensitivity of chicken MM-creatine kinase to trypsin and proteinase-K. , 1985, The International journal of biochemistry.

[27]  H. Wright.  Ovalbumin is an elastase substrate. , 1984, The Journal of biological chemistry.

[28]  M. Morales,et al.  Proteolysis and the domain organization of myosin subfragment 1. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[29]  J. Janin,et al.  Conformational changes in arginine kinase upon ligand binding seen by small‐angle X‐ray scattering , 1983 .

[30]  N. C. Price,et al.  The refolding of denatured rabbit muscle creatine kinase. Search for intermediates in the refolding process and effect of modification at the reactive thiol group on refolding. , 1982, The Biochemical journal.

[31]  N. C. Price,et al.  The effect of limited proteolysis on rabbit muscle creatine kinase. , 1981, The Biochemical journal.

[32]  L. Pradel,et al.  Yeast 3-phosphoglycerate kinase: sulfate and substrate binding, their effect on the conformational state of the enzyme. , 1980, Biochemistry.

[33]  T. Steitz,et al.  Substrate binding closes the cleft between the domains of yeast phosphoglycerate kinase. , 1979, The Journal of biological chemistry.

[34]  T. Steitz,et al.  Space-filling models of kinase clefts and conformation changes. , 1979, Science.

[35]  N. C. Price,et al.  A study of the role of the reactive thiol group of rabbit muscle creatine kinase with a chromophoric reporter group. , 1978, The Biochemical journal.

[36]  E. Milner-White,et al.  Heterogeneity of rabbit muscle creatine kinase and limited proteolysis by proteinase K. , 1977, The Biochemical journal.

[37]  G. L. Kenyon,et al.  Properties of a CH3-blocked creatine kinase with altered catalytic activity. Kinetic consequences of the presence of the blocking group. , 1977, The Journal of biological chemistry.

[38]  G. H. Reed,et al.  Magnetic resonance studies of three forms of creatine kinase. Comparison of the properties of native, CH-S-blocked, and H2NCOCH-blocked enzymes. , 1977, The Journal of biological chemistry.

[39]  E. Milner-White,et al.  Creatine kinase. Modification of the working enzyme. , 1976, The Biochemical journal.

[40]  A. McLaughlin,et al.  Magnetic resonance study of the three-dimensional structure of creatine kinase-substrate complexes. Implications for substrate specificity and catalytic mechanism. , 1976, The Journal of biological chemistry.

[41]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[42]  R. J. Williams,et al.  Nuclear-magnetic-resonance study of the active-site structure of yeast phosphoglycerate kinase. , 1976, European journal of biochemistry.

[43]  R. Haugland.  Conformational flexibility and structure of creatine kinase. , 1975, Journal of supramolecular structure.

[44]  F. Quiocho,et al.  The reaction of creatine kinase with 2-chloromercuri-4-nitrophenol. , 1974, The Journal of biological chemistry.

[45]  G. H. Reed,et al.  Structural changes induced by substrates and anions at the active site of creatine kinase. Electron paramagnetic resonance and nuclear magnetic relaxation rate studies of the manganous complexes. , 1972, The Journal of biological chemistry.

[46]  E. Milner-White,et al.  Inhibition of adenosine 5'-triphosphate-creatine phosphotransferase by substrate-anion complexes. Evidence for the transition-state organization of the catalytic site. , 1971, The Biochemical journal.

[47]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[48]  M. Cohn.  Magnetic resonance studies of enzymesubstrate complexes with paramagnetic probes as illustrated by creatine kinase , 1970, Quarterly Reviews of Biophysics.

[49]  D. Koshland,et al.  The catalytic and regulatory properties of enzymes. , 1968, Annual review of biochemistry.

[50]  W. Cleland,et al.  The statistical analysis of enzyme kinetic data. , 1967, Advances in enzymology and related areas of molecular biology.

[51]  J. Morrison,et al.  The mechanism of the reaction catalysed by adenosine triphosphate-creatine phosphotransferase. , 1965, The Biochemical journal.