Exploring the role of the active site cysteine in human muscle creatine kinase

All known guanidino kinases contain a conserved cysteine residue that interacts with the non-nucleophilic eta1-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 pKa 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 pKa. We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute approximately 1 pH unit, while the presence of Pro284 in the motif lowers the pKa of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pKa of the active site cysteine in WT HMCK and in the P284A, S285A, and C283S/S285C mutants was determined experimentally. The pKa values, although consistently about 0.5 pH unit 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 (Wang et al. (2001) Biochemistry 40, 11698-11705), Cys283 is not responsible for the pKa 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.     

Mutagenesis of two acidic active site residues in human muscle creatine kinase: implications for the catalytic mechanism

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 A 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 gamma-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. The results indicate that of these two residues the Glu232 is the likely catalytic residue while Asp326 likely performs a role in properly aligning substrates for catalysis.     

The active site of creatine kinase. Affinity labeling of cysteine 282 with N-(2,3-epoxypropyl)-N-amidinoglycine.

Epoxycreatine (N-(2,3-epoxypropyl)-N-amidinoglycine) is an affinity label of creatine kinase that irreversibly and completely inactivates the enzyme (Marletta, M. A., and Kenyon, G. L. (1979) J. Biol. Chem. 254, 1879-1886). To identify active site residues of rabbit muscle creatine kinase, the site of modification of it by epoxycreatine has been determined. Separation by high performance liquid chromatography of a tryptic digest of [14C]epoxycreatine-modified creatine kinase yielded two radiolabeled peptides. The larger of these consisted of amino acids Ala-266 through Arg-291 and was labeled with epoxycreatine at Cys-282. Attempts to purify completely the other labeled peptide were not successful; however, it was possible to obtain, by tandem mass spectrometry, a collision-induced dissociation spectrum of it from a mixture of several peptides. This peptide was a fragment (amino acids Val-279 through Arg-291) of the previously identified peptide and was also labeled at Cys-282. Model studies with cysteine and epoxycreatine have demonstrated that opening of the oxirane ring occurs by attack of the cysteine thiolate at the terminal carbon of the epoxide. These results are consistent with previous studies on the base lability of the label; however, a carboxyl group in the active site is not labeled, as had been previously suggested. These results provide evidence that Cys-282 is located in or near the creatine-binding site and will also be important in identifying and delineating the boundaries of the active site of creatine kinase.     

Changing the substrate specificity of creatine kinase from creatine to glycocyamine: evidence for a highly evolved active site

Eight variants of creatine kinase were created to switch the substrate specificity from creatine to glycocyamine using a rational design approach. Changes to creatine kinase involved altering several residues on the flexible loops that fold over the bound substrates including a chimeric replacement of the guanidino specificity loop from glycocyamine kinase into creatine kinase. A maximal 2,000-fold change in substrate specificity was obtained as measured by a ratio of enzymatic efficiency (k(cat)/K(M).K(d)) for creatine vs. glycocyamine. In all cases, a change in specificity was accompanied by a large drop in enzymatic efficiency. This data, combined with evidence from other studies, indicate that substrate specificity in the phosphagen kinase family is obtained by precise alignment of substrates in the active site to maximize k(cat)/K(M).K(d) as opposed to selective molecular recognition of one guanidino substrate over another. A model for the evolution of the dimeric forms of phosphagen kinases is proposed in which these enzymes radiated from a common ancestor that may have possessed a level of catalytic promiscuity. As mutational events occurred leading to greater degrees of substrate specificity, the dimeric phosphagen kinases became evolutionary separated such that the substrate specificity could not be interchanged by a small number of mutations.     

Creatine kinase: a role for arginine-95 in creatine binding and active site organization

Sequence homology analysis reveals that arginine-95 is fully conserved in 29 creatine kinases sequenced to date, but fully conserved as a tyrosine residue in 16 arginine kinases. Site-directed mutants of rabbit muscle creatine kinase (rmCK) were prepared in which R95 was replaced by a tyrosine (R95Y), alanine (R95A), or lysine (R95K). Kinetic analysis of phosphocreatine formation for each purified mutant showed that recombinant native rmCK and all R95 mutants follow a random-order, rapid-equilibrium mechanism. However, we observed no evidence for synergism of substrate binding by the recombinant native enzyme, as reported previously [Maggio et al., (1977) J. Biol. Chem. 252, 1202-1207] for creatine kinase isolated directly from rabbit muscle. The catalytic efficiencies of R95Y and R95A are reduced approximately 3000- and 2000-fold, respectively, compared to native enzyme, but that of R95K is reduced only 30-fold. The major contribution to the reduction of the catalytic efficiency of R95K is a 5-fold reduction in the affinity for creatine. This suggests that while a basic residue is required at position 95 for optimal activity, R95 is not absolutely essential for binding or catalysis in CK. R95Y has a significantly lower affinity for creatine than the native enzyme, but it also displays a somewhat lower affinity for MgATP and 100-fold reduction in k(cat). Interestingly, R95A appears to bind either creatine or MgATP first with affinities similar to those for the native enzyme, but it has a 10-fold lower affinity for the second substrate, suggesting that replacement of R95 by an alanine disrupts the active site organization and reduces the efficiency of formation of the catalytically competent ternary complex.     

An essential tryptophan residue for rabbit muscle creatine kinase

The tryptophan residues in rabbit muscle creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) have been modified by dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide after reversible protection of the reactive SH groups. The modification of two tryptophan residues as measured by spectrophotometric titration leads to complete loss of enzymatic activity. Control experiments show that reversible protection of the reactive SH groups as S-sulfonates followed by reduction results in nearly quantitative recovery of enzyme activity. The presence of a 410 nm absorption maximum and the decrease in fluorescence of the modified enzyme indicate the modification of tryptophan residues. At the same time, SH determinations after reduction of the modified enzyme show that the reagent has not affected the protected SH groups. Quantitative treatment of the data (Tsou, C.-L. (1962) Sci. Sin. 11, 1535 1558) shows that among the tryptophan residues modified, one is essential for its catalytic activity. The presence of substrates partially protects the modification of tryptophan residues as well as the inactivation, suggesting that the essential tryptophan residue is situated at the active site of this enzyme.     

pK values for active site residues of carboxypeptidase A

The phenolic group of active site residue Tyr-248 in carboxypeptidase A has a pKa value of 10.06, as determined from the pH dependence of its rate of nitration by tetranitromethane. The decrease in enzyme activity (kcat/Km) in alkaline solution, characterized by a pKa value of approximately 9.0 (for cobalt carboxypeptidase A), is associated with the protonation state of an imidazole ligand of the active-site metal ion, as indicated by a selective pH dependence of the 1H NMR spectrum of the enzyme. Inhibition of the cobalt-substituted enzyme by 2-(1-carboxy-2-phenylethyl)phenol and its 4,6-dichloro- and 4-phenylazo-derivatives confirms that the decrease in enzyme activity (kcat/Km) in acidic solution, characterized by a pKa value of 5.8, is due to the protonation state of a water molecule bound to the active-site metal ion in the absence of substrate. Changes in the coordination number of the active-site metal ion are seen in its visible absorption spectrum as a consequence of binding of the phenolic inhibitors. Conventional concepts regarding the mechanisms of the enzyme are brought into question.     

Heterogeneity of Escherichia coli-expressed human muscle creatine kinase

Creatine kinase (CK) plays an important role in maintaining a constant ATP:ADP ratio during periods of high energy usage. Elevated levels of CK give an early indication of myocardial infarction. The enzyme has four major isozymes with heterogeneity being observed for each of them. In many cases the source of the heterogeneity is unclear. However, some of the isoforms are known to result from exposure to serum proteases, and analysis of the plasma isoforms provides an estimate of the time of onset of the infarction. Somewhat surprisingly, isoelectric focusing (IEF) experiments provided evidence of heterogeneity in human muscle CK (HMCK) expressed in E. coli. To investigate this further, HMCK was purified to apparent homogeneity utilizing Blue Sepharose affinity chromatography and HiPrep Q anion exchange chromatography. Additional purification on a PBE 94 chromatofocusing column resulted in four fractions, three of which, HMCK I - III, were characterized. The three isoforms are all active and have similar kinetic parameters. They exhibited identical bands on SDS PAGE but different anodal mobility on non-denaturing gels. Modification of C-terminal and/or cysteine residues has been ruled out, and deamidation of asparagine or glutamine residue(s) is proposed to be the cause of isoform formation. In addition each of these isoforms showed a similar four-band pattern on a carrier ampholytes-based IEF gel. Two-dimensional IEF analysis showed that an equilibrium was established between the four bands, suggesting that the four components were unstable and generated only when the protein was subjected to IEF.     

Transition state stabilization by six arginines clustered in the active site of creatine kinase

Six fully conserved arginine residues (R129, R131, R235, R291, R319, and R340) closely grouped in the nucleotide binding site of rabbit muscle creatine kinase (rmCK) were mutated; four to alanine and all six to lysine. Kinetic analyses in the direction of phosphocreatine formation showed that all four alanine mutants led to substantial losses of activity with three (R129A, R131A, and R235A) having no detectable activity. All six lysine mutants retained variable degrees of reduced enzymatic activity. Static quenching of intrinsic tryptophan fluorescence was used to measure the binding constants for MgADP and MgATP. Nucleotide binding was at most only modestly affected by mutation of the arginine residues. Thus, the cluster of arginines seem to be primarily responsible for transition state stabilization which is further supported by the observation that none of the inactive mutants demonstrated the ability to form a transition analogue complex of MgADP.nitrate.creatine as determined by fluorescence quenching assays. As a whole, the results suggest that the most important role these residues play is to properly align the substrates for stabilization of the phosphoryl transfer reaction.     

An (1)H-MRS evaluation of the phosphocreatine/creatine pool (tCr) in human muscle

The human gastrocnemius was examined with and without creatine supplementation under the conditions of rest, ischemic fatigue (IF), and recovery to perturb the pool sizes and equilibrium between phosphocreatine (PCr) and creatine (Cr). (1)H- and (31)P-magnetic resonance spectroscopy (MRS) were used to examine the total creatine (tCr) pool in each of the metabolic states. (31)P-MRS monitored the depletion of the PCr peak during IF to <5% of that at rest. (1)H-MRS focused on the tCr methyl peak at 3.02 ppm (dipolar coupled triplet), at which point it was expected that the triplet peak intensity would be similar both in IF and rest. Initial (1)H-MRS data showed the peak intensity during IF decreased, suggesting a change in tCr pool size. Subsequent studies of transverse relaxation time (T(2)) revealed that this decline was primarily due to a more rapid T(2) decay of the tCr peak in IF (T(2) approximately 40 ms) compared with at rest (T(2) approximately 162 ms). Because Cr is the major contributor to tCr in IF, it is possible that there is a pool of Cr displaying reduced mobility in vivo. Moreover, the residual dipolar coupled triplet observed at rest collapsed into a broad singlet during IF, suggestive of significant changes in the ordered environment experienced at rest for PCr compared with when it is converted to Cr during IF. In addition, these data suggest that in (1)H-MRS studies whose goals include quantitative estimates of tCr pool sizes, standardized metabolic conditions or careful T(2) evaluations will be required.     

Detection of local polarity and conformational changes at the active site of rabbit muscle creatine kinase with a new arginine-specific fluorescent probe

A new polarity-sensitive fluorescent probe, 3-(4-chloro-6-p-glyoxal-phenoxy-1,3,5-triazinylamino)-7-(dimethylamino)-2-methylphenazine (CGTDP), is synthesized for selective labeling of active-site arginine residues. The probe comprises a neutral red moiety as a polarity-sensitive fluorophore and a phenylglyoxal unit as an arginine-specific labeling group. The probe exhibits a sensitive response of shift of fluorescence maximum emission wavelength to solvent polarity only instead of pH or temperature, which leads to the use of the probe in detecting the local polarity and conformational changes of the active site of rabbit muscle creatine kinase (CK) denatured by pH or temperature. The polarity of the active site domain has been first found to correspond to a dielectric constant of about 44, and the conformational change of the active site directly revealed by CGTDP occurs far before that of CK as a whole disclosed by the intrinsic tryptophan fluorescence during acid or thermal denaturation. The present strategy may provide a useful method to detect the local polarity and conformational changes of the active sites of many enzymes that employ arginine residues as anion recognition sites under different denaturation conditions.     

A conserved negatively charged cluster in the active site of creatine kinase is critical for enzymatic activity

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.     

Hydrogen peroxide targets the cysteine at the active site and irreversibly inactivates creatine kinase

In our study, we showed that at a relatively low concentration, H₂O₂ can irreversibly inactivate the human brain type of creatine kinase (HBCK) and that HBCK is inactivated in an H₂O₂ concentration-dependent manner. HBCK is completely inactivated when incubated with 2 mM H₂O₂ for 1 h (pH 8.0, 25 °C). Inactivation of HBCK is a two-stage process with a fast stage (k₁ = 0.050 ± 0.002 min⁻¹) and a slow (k₂ = 0.022 ± 0.003 min⁻¹) stage. HBCK inactivation by H₂O₂ was affected by pH and therefore we determined the pH profile of HBCK inactivation by H₂O₂. H₂O₂-induced inactivation could not be recovered by reducing agents such as dl-dithiothreitol, N-acetyl-l-cysteine, and l-glutathione reduced. When HBCK was treated with DTNB, an enzyme substrate that reacts specifically with active site cysteines, the enzyme became resistant to H₂O₂. HBCK binding to Mg²⁺ATP and creatine can also prevent H₂O₂ inactivation. Intrinsic and 1-anilinonaphthalene-8-sulfonate-binding fluorescence data showed no tertiary structure changes after H₂O₂ treatment. The thiol group content of H₂O₂-treated HBCK was reduced by 13% (approximately 1 thiol group per HBCK dimer, theoretically). For further insight, we performed a simulation of HBCK and H₂O₂ docking that suggested the CYS283 residue could interact with H₂O₂. Considering these results and the asymmetrical structure of HBCK, we propose that H₂O₂ specifically targets the active site cysteine of HBCK to inactivate HBCK, but that substrate-bound HBCK is resistant to H₂O₂. Our findings suggest the existence of a previously unknown negative form of regulation of HBCK via reactive oxygen species.     

Capture of monomeric refolding intermediate of human muscle creatine kinase

Human muscle creatine kinase (CK) is an enzyme that plays an important physiological role in the energy metabolism of humans. It also serves as a typical model for studying refolding of proteins. A study of the refolding and reactivation process of guanidine chloride-denatured human muscle CK is described in the present article. The results show that the refolding process can be divided into fast and slow folding phases and that an aggregation process competes with the proper refolding process at high enzyme concentration and high temperature. An intermediate in the early stage of refolding was captured by specific protein molecules: the molecular chaperonin GroEL and alpha(s)-casein. This intermediate was found to be a monomer, which resembles the "molten globule" state in the CK folding pathway. To our knowledge, this is the first monomeric intermediate captured during refolding of CK. We propose that aggregation is caused by interaction between such monomeric intermediates. Binding of GroEL with this intermediate prevents formation of aggregates by decreasing the concentration of free monomeric intermediates, whereas binding of alpha(s)-casein with this intermediate induces more aggregation.     

Evidence for the participation of Cys558 and Cys559 at the active site of mercuric reductase

Mercuric reductase, with FAD and a reducible disulfide at the active site, catalyzes the two-electron reduction of Hg(II) by NADPH. Addition of reducing equivalents rapidly produces a spectrally distinct EH2 form of the enzyme containing oxidized FAD and reduced active site thiols. Formation of EH2 has previously been reported to require only 2 electrons for reduction of the active site disulfide. We present results of anaerobic titrations of mercuric reductase with NADPH and dithionite showing that the equilibrium conversion of oxidized enzyme to EH2 actually requires 2 equiv of reducing agent or 4 electrons. Kinetic studies conducted both at 4 degrees C and at 25 degrees C indicate that reduction of the active site occurs rapidly, as previously reported [Sahlman, L., & Lindskog, S. (1983) Biochem. Biophys. Res. Commun. 117, 231-237]; this is followed by a slower reduction of another redox group via reaction with the active site. Thiol titrations of denatured Eox and EH2 enzyme forms show that an additional disulfide is the group in communication with the active site. [14C]Iodoacetamide labeling experiments demonstrate that the C-terminal residues, Cys558 and Cys559, are involved in this disulfide. The fluorescence, but not the absorbance, of the enzyme-bound FAD was found to be highly dependent on the redox state of the C-terminal thiols. Thus, Eox with Cys558 and Cys559 as thiols exhibits less than 50% of the fluorescence of Eox where these residues are present as a disulfide, indicating that the thiols remain intimately associated with the active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of creatine kinase isoforms in herring (Clupea harengus) skeletal muscle

It is known that mitochondrial creatine kinase (MtCK) in mammals is always expressed in conjunction with one of the cytosolic forms of creatine kinase (CK), either muscle-type (MM-CK) or brain-type (BB-CK) in tissues of high, sudden energy demand. The two creatine kinase (CK) isoforms were detected in herring (Clupea harengus) skeletal muscle: cytosolic CK and mitochondrial CK (MtCK) that displayed the different electrophoretic mobility. These isoforms differ in molecular weight and some biochemical properties. Isolation and purification procedures allowed to obtain purified enzymes with specific activity of the 206 μmol/min/mg for cytosolic CK and 240 μmol/min/mg for MtCK. Native M_rs of the cytosolic CK and MtCK determined by gel permeation chromatography were 86.000 and 345.000, respectively. The results indicate that one of isoforms found in herring skeletal muscle is a cytosolic dimer and the other one, is a mitochondrial octamer. Octamerization of MtCK is not an advanced feature and also exists in fish. These values correspond well with published values for MtCKs and cytosolic CK isoforms from higher vertebrate classes and even from lower invertebrates.     

The generation of the oxidized form of creatine kinase is a negative regulation on muscle creatine kinase

Muscle creatine kinase (CK) is a crucial enzyme in energy metabolism, and it exists in two forms, the reduced form (R-CK) and the oxidized form (O-CK). In contrast with R-CK, O-CK contained an intrachain disulfide bond in each subunit. Here we explored the properties of O-CK and its regulatory role on muscle CK. The intrachain disulfide bond in O-CK was demonstrated to be formed between Cys(74) and Cys(146) by site-directed mutagenesis. Biophysical analysis indicated that O-CK showed decreased catalytic activity and that it might be structurally unstable. Further assays through guanidine hydrochloride denaturation and proteolysis by trypsin and protease K revealed that the tertiary structure of O-CK was more easily disturbed than that of R-CK. Surprisingly, O-CK, unlike R-CK, cannot interact with the M-line protein myomesin through biosensor assay, indicating that O-CK might have no role in muscle contraction. Through in vitro ubiquitination assay, CK was demonstrated to be a specific substrate of muscle ring finger protein 1 (MURF-1). O-CK can be rapidly ubiquitinated by MURF-1, while R-CK can hardly be ubiquitinated, implying that CK might be degraded by the ATP-ubiquitin-proteasome pathway through the generation of O-CK. The results above were further confirmed by molecular modeling of the structure of O-CK. Therefore, it can be concluded that the generation of O-CK was a negative regulation of R-CK and that O-CK might play essential roles in the molecular turnover of MM-CK.