The tryptophan residues of mitochondrial creatine kinase: Roles of Trp‐223, Trp‐206, and Trp‐264 in active‐site and quaternary structure formation

Groß, Martin; Furter‐Graves, Elizabeth M.; Wallimann, Theo; Eppenberger, Hans M.; Furter, Rolf

doi:10.1002/pro.5560030708

Cited by 67 publications

(68 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the near-UV range (Ն240 nm, Fig. 2B), the three Cotton bands (minima at ϳ280, 290, and 300 nm) characteristic for sMtCK (48) were conserved in all mutant proteins. The octamer/dimer ratio of mutant proteins determined by gel filtration chromatography was even slightly higher than with wild type, providing clear evidence for preservation of the oligomeric state (Fig.…”

Section: Resultsmentioning

confidence: 97%

C-terminal Lysines Determine Phospholipid Interaction of Sarcomeric Mitochondrial Creatine Kinase

Schlattner

Gehring

Vernoux

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

High affinity interaction between octameric mitochondrial creatine kinase (MtCK) and the phospholipid cardiolipin in the inner mitochondrial membrane plays an important role in metabolite channeling between MtCK and inner membrane adenylate translocator, which itself is tightly bound to cardiolipin. Three Cterminal basic residues revealed as putative cardiolipin anchors in the x-ray structures of MtCK and corresponding to lysines in human sarcomeric MtCK (sMtCK) were exchanged by in vitro mutagenesis (K369A/E, K379Q/A/E, K380Q/A/E) to yield double and triple mutants. sMtCK proteins were bacterially expressed, purified to homogeneity, and verified for structural integrity by enzymatic activity, gel filtration chromatography, and CD spectroscopy. Interaction with cardiolipin and other acidic phospholipids was quantitatively analyzed by light scattering, surface plasmon resonance, and fluorescence spectroscopy. All mutant sMtCKs showed a strong decrease in vesicle cross-linking, membrane affinity, binding capacity, membrane ordering capability, and binding-induced changes in protein structure as compared with wild type. These effects did not depend on the nature of the replacing amino acid but on the number of exchanged lysines. They were moderate for Lys-379/Lys-380 double mutants but pronounced for triple mutants, with a 30-fold lower membrane affinity and an entire lack of alterations in protein structure compared with wild-type sMtCK. However, even triple mutants partially maintained an increased order of cardiolipin-containing membranes. Thus, the three Cterminal lysines determine high affinity sMtCK/cardiolipin interaction and its effects on MtCK structure, whereas low level binding and some effect on membrane fluidity depend on other structural components. These results are discussed in regard to MtCK microcompartments and evolution.

show abstract

Section: Resultsmentioning

confidence: 97%

C-terminal Lysines Determine Phospholipid Interaction of Sarcomeric Mitochondrial Creatine Kinase

Schlattner

Gehring

Vernoux

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The transition state analog (TSA) complex (Milner-White & Watts, 1971) was formed by dialysis against MgCI2 (5 mM), ADP (4 mM), KNO, (50 mM), and arginine (20 mM), conditions analogous to those that inhibit CK ( Gross et al, 1994). A reducing agent (DTT, 1 mM) and an antibacterial (sodium azide; 0.05%) were added.…”

Section: Methodsmentioning

confidence: 99%

Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: A model for studying phosphagen kinases

et al. 1997

View full text Add to dashboard Cite

Phosphagen kinases catalyze the reversible transfer of a phosphoryl group between guanidino phosphate compounds and ADP, thereby regenerating ATP during bursts of cellular activity. Large quantities of highly pure arginine kinase (EC 2.7.3.3), the phosphagen kinase present in arthropods, have been isolated from E. coli, into which the cDNA for the horseshoe crab enzyme had been cloned. Purification involves size exclusion and anion exchange chromatographies applied in the denatured and refolded states. The recombinant enzyme has been crystallized as a transition state analog complex. Near complete native diffraction data have been collected to 1.86 A resolution. Substitution of a recombinant source for a natural one, improvement in the purification, and data collection at cry0 temperatures have all yielded significant improvements in diffraction. This reaction and those of the rest of the guanidino (or phosphagen) kinase family, including creatine kinase (CK), enable ATP to be quickly regenerated from storage phosphagens during bursts of cellular activity. In addition to this "temporal" buffering, it has been proposed that these enzymes also function in "spatial" buff-

show abstract

“…This strongly indicated that these modifications are involved in octamer dissociation. In fact, Trp-264 in particular has been identified by site-directed mutagenesis to be crucial for octamer stability (52). To identify which of the two tryptophans in the peptide Gly-263-Arg-271 was modified, we applied postsource decay MALDI-TOF with human sMtCK.…”

Section: Inactivation Of Mtck By Peroxynitrite-the Sensitivity Of Hummentioning

confidence: 99%

Differential Effects of Peroxynitrite on Human Mitochondrial Creatine Kinase Isoenzymes

Wendt

Schlattner

Wallimann

2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Creatine kinase isoenzymes are very susceptible to free radical damage and are inactivated by superoxide radicals and peroxynitrite. In this study, we have analyzed the effects of peroxynitrite on enzymatic activity and octamer stability of the two human mitochondrial isoenzymes (ubiquitous mitochondrial creatine kinase (uMtCK) and sarcomeric mitochondrial creatine kinase (sMtCK)), as well as of chicken sMtCK, and identified the involved residues. Inactivation by peroxynitrite was concentration-dependent and similar for both types of MtCK isoenzymes. Because peroxynitrite did not lower the residual activity of a sMtCK mutant missing the active site cysteine (C278G), oxidation of this residue is sufficient to explain MtCK inactivation. Mass spectrometric analysis confirmed oxidation of Cys-278 and further revealed oxidation of the C-terminal Cys-358, possibly involved in MtCK/membrane interaction. Peroxynitrite also led to concentration-dependent dissociation of MtCK octamers into dimers. In this study, ubiquitous uMtCK was much more stable than sarcomeric sMtCK. Mass spectrometric analysis revealed chemical modifications in peptide Gly-263-Arg-271 located at the dimer/dimer interface, including oxidation of Met-267 and nitration of Trp-268 and/or Trp-264, the latter being a very critical residue for octamer stability. These data demonstrate that peroxynitrite affects the octameric state of MtCK and confirms human sMtCK as the generally more susceptible isoenzyme. The results provide a molecular explanation of how oxidative damage can lead to inactivation and decreased octamer/dimer ratio of MtCK, as seen in neurodegenerative diseases and heart pathology, respectively.Creatine kinases (CK) 1 are key enzymes in energy metabolism by catalyzing the reversible transphosphorylation of creatine by ATP to yield phosphocreatine (PCr) and ADP. The CK system is present in cells with high and fluctuating energy demands, such as skeletal and cardiac muscle, brain, and other neuronal tissues, where a mitochondrial and a cytosolic isoform are coexpressed (for reviews, see Refs.

show abstract

The tryptophan residues of mitochondrial creatine kinase: Roles of Trp‐223, Trp‐206, and Trp‐264 in active‐site and quaternary structure formation

Cited by 67 publications

References 32 publications

C-terminal Lysines Determine Phospholipid Interaction of Sarcomeric Mitochondrial Creatine Kinase

C-terminal Lysines Determine Phospholipid Interaction of Sarcomeric Mitochondrial Creatine Kinase

Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: A model for studying phosphagen kinases

Differential Effects of Peroxynitrite on Human Mitochondrial Creatine Kinase Isoenzymes

Contact Info

Product

Resources

About