Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro (original) (raw)
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Biochemistry and Cell Biology, 2002
The effects of dimethyl sulfoxide (DMSO) on creatine kinase (CK) conformation and enzymatic activity were studied by measuring activity changes, aggregation, and fluorescence spectra. The results showed that at low concentrations (<65% v/v), DMSO had little effect on CK activity and structure. However, higher concentrations of DMSO led to CK inactivation, partial unfolding, and exposure of hydrophobic surfaces and thiol groups. DMSO caused aggregation during CK denaturation. A 75% DMSO concentration induced the most significant aggregation of CK. The CK inactivation and unfolding kinetics were single phase. The unfolding of CK was an irreversible process in the DMSO solutions. The results suggest that to a certain extent, an enzyme can maintain catalytic activity and conformation in water-organic mixture environments. Higher concentrations of DMSO affected the enzyme structure but not its active site. Inactivation occurred along with noticeable conformational change during CK denaturation. The inactivation and unfolding of CK in DMSO solutions differed from other denaturants such as guanidine, urea, and sodium dodecyl sulfate. The exposure of hydrophobic surfaces was a primary reason for the protein aggregation.
Archives of Toxicology
Sulfur mustard (SM) is a chemical warfare agent which use is banned under international law and that has been used recently in Northern Iraq and Syria by the so-called Islamic State. SM induces the alkylation of endogenous proteins like albumin and hemoglobin thus forming covalent adducts that are targeted by bioanalytical methods for the verification of systemic poisoning. We herein report a novel biomarker, namely creatine kinase (CK) B-type, suitable as a local biomarker for SM exposure on the skin. Human and rat skin were proven to contain CK B-type by Western blot analysis. Following exposure to SM ex vivo, the CK-adduct was extracted from homogenates by immunomagnetic separation and proteolyzed afterwards. The cysteine residue Cys282 was found to be alkylated by the SM-specific hydroxyethylthioethyl (HETE)-moiety detected as the biomarker tetrapeptide TC(-HETE)PS. A selective and sensitive micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrom...
Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue
Biochemical Journal, 2000
Protein S-thiolation, the formation of mixed disulphides of cysteine residues in proteins with low-molecular-mass thiols, occurs under conditions associated with oxidative stress and can lead to modification of protein function. In the present study, we examined the site of S-thiolation of the enzyme creatine kinase (CK), an important source of ATP in myocytes. Inactivation of this enzyme is thought to play a critical role in cardiac injury during oxidative stress, such as during reperfusion injury. Reaction of rabbit CK M isoenzyme with GSSG, used to model protein S-thiolation, was found to result in enzyme inactivation that could be reversed by GSH or dithiothreitol. Measurement of GSH that is released during the thiolation reaction indicated that the maximum extent of CK thiolation was approx. 1 mol of GSH\mol of protein, suggesting thiolation on one reactive cysteine residue. Accordingly, matrix-assisted laser-desorption
Methionine329in human serum albumin: A novel target for alkylation by sulfur mustard
Drug Testing and Analysis, 2018
Exposure to the vesicant sulfur mustard (SM) may lead to erythema and blistering. Toxicity of SM is hypothesized to be due to the alkylation of DNA bases and nucleophilic amino acid side chains in proteins (adducts) by forming the hydroxyethylthioethyl (HETE) moiety. Despite of its prohibition by the chemical weapons convention, SM still represents a serious threat to military personnel and civilians. Therefore, development and improvement of forensic analytical methods for the verification of SM exposure is of high interest. Protein adducts have been shown to be highly suitable and beneficial biomarkers of poisoning. Herein we present methionine 329 in human serum albumin (HSA) as a novel target of SM forming a HETE-methionyl sulfonium ion. The alkylated tetrapeptide LeuGlyMet 329 (-HETE)Phe, LGM(-HETE)F, was detected after pepsin mediated proteolysis and subsequent analysis by microbore liquid chromatography-electrospray ionization highresolution tandem-mass spectrometry. Compound identity was confirmed by a synthetic reference. Proteolysis conditions for HSA were optimized towards maximum yield of LGM(-HETE)F and its limit of identification (32.3 nM SM in serum) was similar to those of the established HSA-derived biomarkers HETE-CysPro and HETE-CysProPhe (15.6 nM SM in serum). Stability of the alkylated Met 329 in vitro and in vivo was limited to 5 days making this modification a beneficial short-time biomarker. Furthermore, it was found that the HETE-methionyl sulfonium ion can transfer its HETE moiety to the side chain of cysteine and glutamic acid as well as to the N-terminus of peptides and proteins in vitro thus revealing novel insights into the molecular toxicity of SM.
Rabbit Muscle Creatine Kinase: Consequences of the Mutagenesis of Conserved Histidine Residues †
Biochemistry, 1996
Creatine kinase (CK; EC 2.7.3.2) catalyzes the reversible conversion of creatine and MgATP to phosphocreatine and MgADP. In the absence of an X-ray crystal structure, we have used the sequence homology of creatine kinases and other guanidino kinases from a variety of sources to identify the conserved histidine residues in rabbit muscle CK, as well as to try to pinpoint a reactive histidine that has been implicated in the active site. This residue has been proposed to act as a general acid/base catalyst assisting in the phosphoryl transfer mechanism ) Biochemistry 20, 1204-1210. There are 17 histidine residues in rabbit muscle CK, and of these, only five have been conserved in all guanidino kinase sequences published to date [Mühlebach et al. (1994) Mol. Cell. Biochem. 133, 245-62]. In rabbit muscle CK, these residues are H96, H105, H190, H233, and H295. We have carried out sitespecific mutagenesis of these five histidine residues, replacing each with an asparagine. Each of these mutants exhibited enzymatic activity but to varying degrees. , and H233N mutants displayed specific activities similar to that of the wild-type enzyme. H96N has high activity, but appears to be quite unstable, losing catalytic activity upon cell lysis by sonication and/or chromatographic steps involved in purification. H295N shows a significantly reduced catalytic activity relative to the native enzyme, due to marked decreases in k cat and the affinities for both substrates. Each of the five mutants is inactivated by diethyl pyrocarbonate (DEP), and inactivation is reversible upon incubation with hydroxylamine. However, only H295N shows a dramatically reduced rate of inactivation relative to native CK, consistent with H295 being the residue modified by DEP in the native enzyme. These intriguing results indicate that four of the conserved histidines (H96, H105, H295, and H233) are not essential for activity, and while H295 may be at the active site of CK, it is unlikely to play the role of a general acid/base catalyst. † LB/Amp, Luria-Bertani medium containing 50 µg/mL ampicillin or its analog, carbenicillin; DEP, diethyl pyrocarbonate; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); TNB-derivatized CK, creatine kinase in which the reactive thiol of Cys282 has been reversibly modified by DTNB; H96N, histidine at position 96 of the rabbit muscle CK that has been replaced by asparagine.
Free radical induced inactivation of creatine kinase: sites of interaction, protection, and recovery
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2000
The study aims at a clarification of the oxidative damage of creatine kinase isoenzymes by X-ray-induced water radiolysis. The radical species generated by this method (under appropriate conditions) are similar to those discussed in the context of mitochondrial energy metabolism. The decay of the enzyme activity is accompanied by a strong decrease of the number of accessible SH groups and by a reduction of the endogenous tryptophan fluorescence. Free radical effects are diminished if irradiation is carried out in the presence of 2-mercaptoethanol. Partial recovery of the activity (repair) is observed if 2-mercaptoethanol is added after irradiation. The experiments suggest a twofold importance of thiol reagents (RSH): to reduce the concentration of free radicals by scavenger reactions and to modify the inactivation mechanism in such a way that efficient repair of enzyme damage may be achieved. Cysteine 282 of MM-CK (Cys-278 in the case of Mi-CK) seems to play a crucial role in this respect. Blockage of the SH group of cysteine 282 by oxidized glutathione effectively protects the enzyme against inactivation by NO c 2 radicals. In the absence of nitrogen dioxide and of thiol reagents, however, inactivation seems to proceed via a less specific mechanism involving additional targets of the enzyme. ß
Clinica Chimica Acta, 1978
We investigated the effects of sulfhydryl compounds on the stability of creatine kinase (CK) in unfrozen human serum and found that both P-mercaptoethanol and N-acetylcysteine led to accelerated loss of the endogenous serum enzyme activity. This is in contrast to the results of others who have either studied the stability of exogenous enzyme added to human serum or studied endogenous enzyme in frozen serum. The addition of cation chelators to serum markedly improved the stability of the endogenous CK activity. The enhanced stability was independent of chelation of calcium, iron, manganese, copper, or zinc. In addition, cation chelators caused a 16% increase in the CK activity of fresh samples. This latter effect was independent of the activation of CK by BME and could be accounted for by chelation of calcium ions during the assay. The data suggest that addition of cation chelators prior to storage may be useful in enhancing the stability of CK in serum whereas sulfhydryl compounds should be added prior to assay rather than prior to storage.
The Journal of biological chemistry, 1977
Steady state kinetic parameters for rabbit muscle creatine kinase (EC 2.7.3.2) and this enzyme stoichiometrically blocked at the iodoacetamide-sensitive cysteinyl residue with a CH3S-group have been measured at 30+/-0.1 degrees, pH 9.00, using Mg(II) as the required metal ion cofactor. The double reciprocal plots for the CH3S-blocked enzyme with MgATP as the variable substrate are biphasic, each curve showing a break at approximately 1.9 mM MgATP, and suggest the possibility of negative cooperativity in metal-nucleotide binding. Furthermore, extrapolated lines at high MgATP concentrations intersect on the abscissa, indicating loss of synergism in binding of substrates. In contrast, observed Michaelis constants for creatine are, within experimental error, the same for both native and blocked enzymes. The maximal velocity of the CH3S-blocked enzyme is found to be 28.1% of the value of the native enzyme. Product inhibition patterns for both native and blocked enzyme are also compared. ...
Exploring the Role of the Active Site Cysteine in Human Muscle Creatine Kinase
Biochemistry, 2006
All known guanidino kinases contain a conserved cysteine residue that interacts with the nonnucleophilic η 1 -nitrogen of the guanidino substrate. Site-directed mutagenesis studies have shown that this cysteine is important, but not essential for activity. In human muscle creatine kinase (HMCK) this residue, Cys283, forms part of a conserved cysteine-proline-serine (CPS) motif and has a pK a about 3 pH units below that of a regular cysteine residue. Here we employ a computational approach to predict the contribution of residues in this motif to the unusually low cysteine pK a . We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute ~1 pH unit, while the presence of Pro284 in the motif lowers the pK a of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pK a of the active site cysteine in WT HMCK and in the P284A, S285A and C283S/S285C mutants was determined experimentally. The pK a values, although consistently about 0.5 pH units lower, were in broad agreement with those predicted. The effect of each of these mutations on the pH-rate profile was also examined. The results show conclusively that, contrary to a previous report Biochemistry 40, 11698-11705), Cys283 is NOT responsible for the pK a of 5.4 observed in the WT V/K creatine pH profile. Finally we use molecular dynamics simulations to demonstrate that, in order to maintain the linear alignment necessary for associative inline transfer of a phosphoryl group, Cys283 needs to be ionized.