Subunit-selective chemical modifications of creatine kinase. Evidence for asymmetrical association of the subunits (original) (raw)
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Creatine kinase: The reactive cysteine is required for synergism but is nonessential for catalysis
Biochemistry, 1993
Chemical modification of rabbit muscle creatine kinase (CK) with thiol-specific reagents led to partial or complete inactivation of the enzyme. Using site-directed mutagenesis, we have substituted the corresponding reactive Cys278 in the chicken cardiac mitochondrial creatine kinase (Mib-CK) with either glycine, serine, alanine, asparagine, or aspartate. The resulting mutant Mib-CK enzymes showed qualitatively similar changes in their enzymatic properties. In both directions of the CK reaction, a shift of the pH optimum to lower values was observed. Mutant Mib-CKs were severalfold more sensitive to inhibition by free ADP in the reverse reaction (ATP synthesis) and to free ATP in the forward reaction (phosphocreatine synthesis). With the exception of C278D, all mutant enzymes were specifically activated by chloride and bromide anions. C278D and wild-type Mib-CK were significantly inhibited under the same conditions. At low chloride concentrations, the V,,, of C278D was about 12-fold higher than that of C278N. Thus, Cys278 probably provides a negative charge which is directly or indirectly involved in maximizing CK activity. Under near-optimal conditions in the reverse reaction, mutants C278G and C278S showed about an 1 l-fold increase inKm(PCr), but only 1.7-and 2.8-fold reductions in V-, respectively, compared to wild-type Mib-CK. Thus, the reactive cysteine clearly is not essential for catalysis. For rabbit muscle CK, substrate binding had been shown to be synergistic (Le., Kd > K,,,). We confirmed this finding for wild-type Mib-CK by determining the Kd and K m values for both substrates in the forward reaction. Analysis of these constants for the two mutant enzymes C278G and C278S showed that the reactive cysteine (1) is not directly involved in binding either substrate (&values for mutants were not dramatically changed compared to the wild type) and is necessary for synergistic substrate binding (Kd values for mutants were smaller than the corresponding Km values). These results suggest that the reactive cysteine is necessary to confer conformational changes upon substrate binding and support the proposal that this residue has a role in shaping the active site, possibly by acting as a hinge between the two substrate binding sites. * Corresponding author. Telephone (01) 377 3447, FAX (01) 371 2894. 1 Abbreviations: Ac, acetate anion; CK, creatine kinase; B-CK, braintype C K M-CK, muscle-type CK; Mi-CK, mitochondrial-type CK isoforms; Mib-CK, basic, "sarcomeric" Mi-CK isoform; PCr, phosphocreatine; Cr, creatine. Mutants with amino acid substitutions are denoted in the standard one-letter code by the wild-type residue and numbered position within the sequence, followed by the amino acid substitution. Thus, C278G Mib-CK is a mutant enzyme with a glycine residue at position 278 instead of thecysteine. Kd refers to the dissociation constant of the reaction E + A -EA or E + B -EB, and K m refers to the Michaelis-Menten constant of the reaction EA + B -EAB or EB + A -EAB, whereby A and B represent either ADP and PCr or ATP and Cr, respectively. Forward reaction is PCr synthesis; reverse reaction is ATP synthesis.
Why is creatine kinase a dimer? Evidence for cooperativity between the two subunits
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2000
The dimeric chicken brain type isoenzyme of creatine kinase (BB-CK) was mutated by a C283S amino acid exchange in the catalytic site to produce a basically inactive dimer (B*B*-CK). The mutated enzyme showed a residual activity of about 4% compared to the wild-type, whereas substrate binding parameters were not altered. The inactivated dimer was hybridized with native dimeric muscle enzyme (MM-CK) to produce a partially inactivated MB*-CK heterodimeric hybrid and also to a his-tagged BB-CK (hBhB-CK) resulting in a partially inactive hBB*-CK homodimer. The generated hybrids were purified by chromatography. The V max and substrate binding parameters K m and K d were determined for both directions of the CK reaction and compared to the parameters of the wild-type enzymes (MM-, BB-, hBhB-, MB-CK). In the direction of ATP synthesis (reverse reaction), the MB*-and hBB*-CK hybrids showed a decrease of V max to 34% and 32%, respectively, compared to the unmodified wild-type isoform. The inactivation of a single subunit in MB*-CK led to an increase in the K d value resulting in an significant substrate synergism, not seen with the MB-CK wild-type enzyme. In the direction of phosphocreatine synthesis (forward reaction), the modified hybrids showed a decrease of V max to 50% of the wild-type enzymes and no significant alterations of the K m and K d parameters. These results strongly suggest an enzymatic cooperativity of the two subunits in the reverse reaction but independent catalytic function in the forward reaction. ß 2000 Elsevier Science B.V. All rights reserved.
Journal of Biological Chemistry, 2000
Creatine kinase catalyzes the reversible transphosphorylation of creatine by MgATP. From the sequence homology and the molecular structure of creatine kinase isoenzymes, we have identified several highly conserved residues with a potential function in the active site: a negatively charged cluster (Glu 226 , Glu 227 , Asp 228 ) and a serine (Ser 280 ). Mutant proteins E226Q, E226L, E227Q, E227L, D228N, and S280A/S280D of human sarcomeric mitochondrial creatine kinase were generated by in vitro mutagenesis, expressed in Escherichia coli, and purified to homogeneity. Their overall structural integrity was confirmed by CD spectroscopy and gel filtration chromatography. The enzymatic activity of all proteins mutated in the negatively charged cluster was extremely low (0.002-0.4% of wild type) and showed apparent Michaelis constants (K m ) similar to wild type, suggesting that most of the residual activity may be attributed to wild-type revertants. Mutations of Ser 280 led to higher residual activities and altered K m values; S280A showed an increase of K m for phosphocreatine (65-fold), creatine (6-fold), and ATP (6-fold); S280D showed a decrease of K m for creatine 6-fold). These results, together with the transition state structure of the homologous arginine kinase (Zhou, G., Somasundaram, T., Blanc, E., Parthasarathy G., Ellington, W. R., and Chapman, M. S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8449 -8454), strongly suggest a critical role of Glu 226 , Glu 227 , and Asp 228 in substrate binding and catalysis and point to Glu 227 as a catalytic base.
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.
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
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. ...
Biochemistry, 2001
Creatine kinase (CK) catalyzes the reversible phosphorylation of the guanidine substrate, creatine, by MgATP. Although several X-ray crystal structures of various isoforms of creatine kinase have been published, the detailed catalytic mechanism remains unresolved. A crystal structure of the CK homologue, arginine kinase (AK), complexed with the transition-state analogue (arginine-nitrate-ADP), has revealed two carboxylate amino acid residues (Glu225 and Glu314) within 2.8 Å of the proposed transphosphorylation site. These two residues are the putative catalytic groups that may promote nucleophilic attack by the guanidine amino group on the γ-phosphate of ATP. From primary sequence alignments of arginine kinases and creatine kinases, we have identified two homologous creatine kinase acidic amino acid residues (Glu232 and Asp326), and these were targeted for examination of their potential roles in the CK mechanism. Using site-directed mutagenesis, we have made several substitutions at these two positions.
SpringerReference
The purpose of this investigation was to develop a simple colorimetric method for creatine kinase (CK). The new method is based on the reaction of creatine, formed enzymatically from creatine phosphate and ADP, with different glyoxal compounds. Hydrated glyoxals, such as para-nitrophenyl, 2-thiophene, 4biphenyl, 4, 4'-biphenyl, a-naphthyl, f3-naphthyl, para-chlorophenyl, and styryl were synthesized and allowed to react with creatine. Among the glyoxals, the 2thiophene derivative was the best in terms of the stability and intensity of the colored complex which was produced under mild alkaline conditions. The complex absorbed maximally at 460 nm with an extinction coefficient of 1.56x 1 04 M-I Cm-I. This reagent was used to determine CK in the sera of normal human beings and patients with myocardial infarction. The results obtained were in agreement with those obtained by another available method for CK. However, this new method is simple, less time consuming and employs a single reagent for color development. Such a simple method might be of value in clinical laboratories with little access to sophisticated instruments such as auto analyzers and spectropho tometers.