A Conserved Negatively Charged Cluster in the Active Site of Creatine Kinase Is Critical for Enzymatic Activity

Abstract

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 (Glu226, Glu227, Asp228) and a serine (Ser280). 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 Ser280 led to higher residual activities and altered K m values; S280A showed an increase ofK m for phosphocreatine (65-fold), creatine (6-fold), and ATP (6-fold); S280D showed a decrease ofK 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 Glu226, Glu227, and Asp228in substrate binding and catalysis and point to Glu227 as a catalytic base. 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 (Glu226, Glu227, Asp228) and a serine (Ser280). 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 Ser280 led to higher residual activities and altered K m values; S280A showed an increase ofK m for phosphocreatine (65-fold), creatine (6-fold), and ATP (6-fold); S280D showed a decrease ofK 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 Glu226, Glu227, and Asp228in substrate binding and catalysis and point to Glu227 as a catalytic base. creatine kinase phosphocreatine creatine arginine kinase ubiquitous and sarcomeric mitochondrial CK, respectively 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol transition state analog complex Creatine kinase (CK;1 EC2.7.3.2) catalyzes the interconversion of phosphocreatine (PCr) and ADP with ATP and creatine (Cr). The enzyme occurs as a family of tissue-specific isoenzymes, comprising dimeric cytosolic CK (MM-, MB-, and BB-CK) and mainly octameric mitochondrial CK (sMtCK and uMtCK), which can dissociate into dimers. These CK isoenzymes, together with easily diffusable Cr and PCr, maintain a unique cellular energy buffer and energy transport system, the CK/PCr circuit (for reviews see Refs.1Wallimann T. Wyss M. Brdicka D. Nicolay K. Eppenberger H.M. Biochem. J. 1992; 281: 21-40Crossref PubMed Scopus (1599) Google Scholar and 2Schlattner U. Forstner M. Eder M. Stachowiak O. Fritz-Wolf K. Wallimann T. Mol. Cell. Biochem. 1998; 184: 125-140Crossref PubMed Google Scholar). Thus, the CK system plays a key role in energy metabolism of cells and tissues with high or fluctuating energy requirements like muscle or brain. Lately, the molecular structures of all four homo-oligomeric CK isoenzymes have been solved (3Fritz-Wolf K. Schnyder T. Wallimann T. Kabsch W. Nature. 1996; 381: 341-345Crossref PubMed Scopus (261) Google Scholar, 4Rao J.K. Bujacz G. Wlodawer A. FEBS Lett. 1998; 439: 133-137Crossref PubMed Scopus (140) Google Scholar, 5Eder M. Schlattner U. Becker A. Wallimann T. Kabsch W. Fritz-Wolf K. Prot. Sci. 1999; 8: 2258-2269Crossref PubMed Scopus (97) Google Scholar, 6Eder M. Fritz-Wolf K. Kabsch W. Wallimann T. Schlattner U. Proteins. 1999; 39: 216-225Crossref Google Scholar), as well as the transition state structure of monomeric arginine kinase (AK) from horseshoe crab (7Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8449-8454Crossref PubMed Scopus (234) Google Scholar). Both CK and AK belong to the larger guanidino kinase family. These new data allow a fresh look on amino acid residues with a critical role in substrate binding and catalysis. The catalytic mechanism of CK has been studied extensively by numerous different techniques, yielding detailed information on kinetic and mechanistic aspects of the transphosphorylation reaction (8Kenyon G.L. Reed G.H. Adv. Enzymol. 1983; 54: 367-426PubMed Google Scholar). It has been well documented that the γ-phosphoryl group is transferred via an associative in-line mechanism (9Milner-White E.J. Watts D.C. Biochem. J. 1971; 122: 727-740Crossref PubMed Scopus (137) Google Scholar, 10McLaughlin A.C. Cohn M. Kenyon G.L. J. Biol. Chem. 1972; 247: 4382-4388Abstract Full Text PDF PubMed Google Scholar, 11Hansen D.E. Knowles J.R. J. Biol. Chem. 1981; 256: 5967-5969Abstract Full Text PDF PubMed Google Scholar). At pH 8 and above, the CK reaction follows a rapid equilibrium random mechanism in both directions (12Morrison J.F. Cleland W.W. J. Biol. Chem. 1966; 241: 673-683Abstract Full Text PDF PubMed Google Scholar), whereas at pH 7, the kinetic mechanism is random only in the reverse direction (ATP synthesis) and equilibrium ordered, with ATP adding before Cr, in the forward direction (PCr synthesis) (13Schimerlik M.I. Cleland W.W. J. Biol. Chem. 1973; 248: 8418-8423Abstract Full Text PDF PubMed Google Scholar). By contrast, our knowledge about specific amino acid residues involved in substrate binding and catalysis is scarce. Especially the guanidino substrate-binding site of CK has not yet been characterized, because of the lack of specific mutants or a CK crystal structure containing creatine. Mutation of the highly reactive cysteine 278 (sMtCK numbering) and tryptophan 223 located near the active site led to severely decreased enzymatic activity (14Furter R. Furter-Graves E.M. Wallimann T. Biochemistry. 1993; 32: 7022-7029Crossref PubMed Scopus (111) Google Scholar, 15Gross M. Furter-Graves E.M. Wallimann T. Eppenberger H.M. Furter R. Prot. Sci. 1994; 3: 1058-1068Crossref PubMed Scopus (67) Google Scholar). C278 was implicated in substrate synergism (14Furter R. Furter-Graves E.M. Wallimann T. Biochemistry. 1993; 32: 7022-7029Crossref PubMed Scopus (111) Google Scholar) and may interact with the creatine substrate (7Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8449-8454Crossref PubMed Scopus (234) Google Scholar). Several arginines were identified to interact electrostatically with the negatively charged phosphate groups of the nucleotide (16Wood T.D. Guan Z. Borders Jr., C.L. Chen L.H. Kenyon G.M. Lafferty F.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3362-3365Crossref PubMed Scopus (44) Google Scholar). From earlier work, a histidine residue was proposed to act as an acid base catalyst in the transphosphorylation reaction (17Cook P.F. Kenyon G.L. Cleland W.W. Biochemistry. 1981; 20: 1204-1210Crossref PubMed Scopus (90) Google Scholar). However, recent site-directed mutagenesis experiments (18Chen L.H. Borders Jr., C.L. Vasquez J.R. Kenyon G.L. Biochemistry. 1996; 35: 7895-7902Crossref PubMed Scopus (44) Google Scholar, 19Forstner M. Müller A. Stolz M. Wallimann T. Prot. Sci. 1997; 6: 331-339Crossref PubMed Scopus (27) Google Scholar), as well as the AK structure (7Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8449-8454Crossref PubMed Scopus (234) Google Scholar), have clearly demonstrated that none of the conserved histidines is able to provide this function. It was speculated that CK might act as a “conzyme” without catalytic residue by just bringing the substrates into a close, favorable alignment (20Stroud R.M. Nat. Struct. Biol. 1996; 3: 567-569Crossref PubMed Scopus (24) Google Scholar). Alternatively, other residues may be involved to draw away partial positive charge from the reactive guanidinium-Nη2 or even to act as a catalytic base. From the AK transition state structure, two glutamates were proposed as new candidates for acid base catalysis (7Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8449-8454Crossref PubMed Scopus (234) Google Scholar). In analogy to ATP hydrolysis at the myosin motor domain (21Fisher A.J. Smith C.A. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (636) Google Scholar), also the γ-phosphate of ATP itself could act as a catalytic base, assisted by a serine that participates in hydrogen exchange by providing an energetically favorable geometry. In the present study, we have analyzed the available sequence and structural information for CK and AK to look for residues with a putative role in the active site of CK. Using site-directed mutagenesis with the recently characterized human sMtCK isoenzyme (22Schlattner U. Wallimann T. J. Biol. Chem. 2000; 275: 17314-17320Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), we could identify four highly conserved residues, including a negatively charged cluster and a serine residue, which are critical for substrate binding and catalytic mechanism. The sequence of mature human sMtCK (GenBankTMJ05401) has been cloned into a pET-derived expression vector (22Schlattner U. Wallimann T. J. Biol. Chem. 2000; 275: 17314-17320Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 23Furter R. Kaldis P. Furter-Graves E. Schnyder T. Eppenberger H.M. Wallimann T. Biochem. J. 1992; 288: 771-775Crossref PubMed Scopus (44) Google Scholar). Site-directed mutagenesis by inverse polymerase chain reaction was carried out on a Hybaid Omn-E Thermal Cycler (MWG-Biotech, Münchenstein, Switzerland), using 10 ng of double-stranded DNA (entire plasmid vector), 15 pmol of each oligonucleotide primer, and Pfu DNA polymerase (Stratagene, Zürich, Switzerland). The latter was added to the reaction mixture at 95 °C (“hot start”) followed by 30 polymerase chain reaction cycles (95 °C, 0.5 min; 55 °C, 0.5 min; 72 °C, 6.5 min). All primers were 5′-phosphorylated for subsequent circularization of gel-purified polymerase chain reaction products with were into E. using K. J. T. R. Smith K. in and Scholar) and with the chain S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) to in vitro mutagenesis and the of random primers with the are S280D S280A of E. were at °C in expression of at about by the of was for about was from by the of of to sMtCK was from at an of in buffer and sMtCK was purified by high with the Switzerland), with buffer pH sMtCK in a at about and were and with to about by gel filtration involved a and was and with gel filtration buffer pH and at a of The as by gel was to Biochem. PubMed Scopus Google Scholar), using as a CK activity and kinetic constants were with a enzyme Wallimann T. D.C. Eppenberger H.M. J. Biol. PubMed Scopus Google Scholar). ATP was by and to using PCr, and in m pH The of was by kinase and to using and in pH in the state of were followed at in a at All substrates and for activity were from Switzerland). in the pH of was to pH M. J. Wallimann T. 1992; PubMed Scopus Google Scholar). In the pH of enzymatic activity was with pH buffer and from pH to together with activities of constants (K m) were by to different of each substrate at a of the the of kinetic constants was by of the reaction to the of octameric and dimeric sMtCK by gel filtration and of sMtCK by with transition state analog complex (9Milner-White E.J. Watts D.C. Biochem. J. 1971; 122: 727-740Crossref PubMed Scopus (137) Google was carried out as in U. Wallimann T. J. Biol. Chem. 2000; 275: 17314-17320Abstract Full Text Full Text PDF PubMed Scopus (91) Google and near CD of human sMtCK wild-type and were on a at °C and using a with a sMtCK in phosphate was in this buffer to about and highly conserved of the CK sequence and M. T. Wallimann T. Wyss M. Mol. Cell. Biochem. 1994; Scopus Google of the putative active site (3Fritz-Wolf K. Schnyder T. Wallimann T. Kabsch W. Nature. 1996; 381: 341-345Crossref PubMed Scopus (261) Google Scholar) and most residues involved in substrate binding or catalysis (14Furter R. Furter-Graves E.M. Wallimann T. Biochemistry. 1993; 32: 7022-7029Crossref PubMed Scopus (111) Google Scholar) or M. Furter-Graves E.M. Wallimann T. Eppenberger H.M. Furter R. Prot. Sci. 1994; 3: 1058-1068Crossref PubMed Scopus (67) Google Scholar), sMtCK The only negatively charged amino in this a cluster (Glu226, Glu227, and Asp228) that is conserved all creatine and even the larger guanidino kinase family M. T. Wallimann T. Wyss M. Mol. Cell. Biochem. 1994; Scopus Google Scholar). It is located near the γ-phosphate of ATP in sMtCK (3Fritz-Wolf K. Schnyder T. Wallimann T. Kabsch W. Nature. 1996; 381: 341-345Crossref PubMed Scopus (261) Google and may be well for or binding guanidino The Glu227 of AK with the guanidino group (7Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8449-8454Crossref PubMed Scopus (234) Google Scholar). highly conserved residue, which is by in guanidino M. T. Wallimann T. Wyss M. Mol. Cell. Biochem. 1994; Scopus Google Scholar), has to the γ-phosphate of ATP and the active site cysteine 278 (3Fritz-Wolf K. Schnyder T. Wallimann T. Kabsch W. Nature. 1996; 381: 341-345Crossref PubMed Scopus (261) Google It be a for catalysis (21Fisher A.J. Smith C.A. Thoden J.B. Smith R. Sutoh K. Holden H.M. Rayment I. Biochemistry. 1995; 34: 8960-8972Crossref PubMed Scopus (636) Google Scholar). amino acid mutants of human sMtCK were generated by site-directed mutagenesis and by DNA Glu226, Glu227, and were with E227Q, and or amino E227L, and Ser280 was by or for all mutants could be expressed in E. not to like wild-type we low at pH 6.5 for a at led to a of of as from gel not by exchange and gel filtration about of of which is for the wild-type All purified sMtCK proteins and active at °C, suggesting that and were not CD of all mutants were to of wild-type the overall structural integrity CD in the near for the only to the wild-type enzyme with a of the for CK M. Furter-Graves E.M. Wallimann T. Eppenberger H.M. Furter R. Prot. Sci. 1994; 3: 1058-1068Crossref PubMed Scopus (67) Google Scholar). in the of residues were and because of by the charge The of proteins at was at is to wild-type Schlattner and T. that the on the state of human sMtCK of of sMtCK before and for 72 at as from gel filtration were to in pH in a new of sMtCK before and for 72 at as from gel filtration were to in pH activities and kinetic for wild-type and were in the forward reaction (PCr at pH as well as in the reverse reaction (ATP at pH by a enzyme E226Q, E226L, E227Q, residual catalytic activities as as of wild-type activity in the forward as well as in the reverse reaction enzymatic activities of and S280D were higher extremely low as with wild-type human sMtCK of wild-type S280A showed the residual in the reverse reaction of wild-type could that of the residues the pH of the CK because none of the proteins showed residual activity at other pH for the not (K m) of each were for all substrates Mutations of residues led to K m to wild-type K m for PCr, was By contrast, S280A showed a increase ofK m together with a increase ofK m and K m whereas S280D a decrease of K m (Cr). of residues may the of we also the at nucleotide was in as with wild-type a in our not constants of the (K could not be because of both substrates residual activities at or the of the enzyme activities of human sMtCK Cr ATP ADP pH ADP Cr pH of enzyme activity is to of ATP or at of enzyme activity is to of ATP or at enzyme activities are from at using a enzyme (22Schlattner U. Wallimann T. J. Biol. Chem. 2000; 275: 17314-17320Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Cr ATP ADP pH ADP Cr pH of enzyme activity is to of ATP or at in a new kinetic constants of human sMtCK m m m m were using a enzyme (22Schlattner U. Wallimann T. J. Biol. Chem. 2000; 275: 17314-17320Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). constants were by the data from at to the in a new enzyme activities are from at using a enzyme (22Schlattner U. Wallimann T. J. Biol. Chem. 2000; 275: 17314-17320Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The were using a enzyme (22Schlattner U. Wallimann T. J. Biol. Chem. 2000; 275: 17314-17320Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). constants were by the data from at to the are to dissociate into the of substrates that a M. Wallimann T. Biochemistry. 1993; 32: PubMed Scopus Google Scholar, O. D. D. PubMed Scopus Google Scholar). is because of by the binding of M. M. P. Wallimann T. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) that the of octameric sMtCK for was severely in the of and mutants have a decreased for substrates or is to the U. Forstner M. Eder M. Stachowiak O. Fritz-Wolf K. Wallimann T. Mol. Cell. Biochem. 1998; 184: 125-140Crossref PubMed Google Scholar). Using a site-directed mutagenesis we have identified four residues in the active site of human sMtCK that are for substrate binding and Glu226, Glu227, and Ser280 (sMtCK Mutation of residues proteins with enzymatic activities and of together with a well conserved overall The latter is by binding to exchange at °C, near and CD and The residual enzymatic activity of the CK mutants could not be by or the pH in the can binding or a in pH as for the strongly enzymatic as was in cysteine mutants (14Furter R. Furter-Graves E.M. Wallimann T. Biochemistry. 1993; 32: 7022-7029Crossref PubMed Scopus (111) Google Scholar). Mutations of residues and Glu227 in the negatively charged cluster showed the most decrease of enzymatic activity by of yielding of were an of higher with wild-type These low residual enzymatic activities of may from that have the wild-type residue by (20Stroud R.M. Nat. Struct. Biol. 1996; 3: 567-569Crossref PubMed Scopus (24) Google Scholar, Biol. 1994; Google Scholar). we have by E. in our the in E. to is the of expression and could for residual enzymatic activities as high as of wild mutants of the tryptophan 223 and cysteine 278 showed higher residual activities in the of and at respectively (14Furter R. Furter-Graves E.M. Wallimann T. Biochemistry. 1993; 32: 7022-7029Crossref PubMed Scopus (111) Google Scholar, 15Gross M. Furter-Graves E.M. Wallimann T. Eppenberger H.M. Furter R. Prot. Sci. 1994; 3: 1058-1068Crossref PubMed Scopus (67) Google Scholar). Mutations of Ser280 also a active with S280A of was speculated that near or at the active site of CK not and that CK is a enzyme C.A. PubMed Scopus Google Scholar, G.L. Nature. 1996; 381: PubMed Scopus Google Scholar). data clearly that this is not the and strongly suggest a critical role of Glu226, Glu227, and in substrate binding catalysis of CK. substrate binding is at of the that we have is clearly by the binding of proteins to which a and the of the other apparent (K m) of in the negatively charged cluster were with may the of wild-type to the low residual activity of The is different for serine higher residual enzymatic activities and apparent K m different from wild-type the of a active In the in m were on the amino m for Cr, and in S280A decreased for Cr in The recently transition state structure of AK (7Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8449-8454Crossref PubMed Scopus (234) Google Scholar) a of the mutated CK the overall of AK follows that of all CK structures (3Fritz-Wolf K. Schnyder T. Wallimann T. Kabsch W. Nature. 1996; 381: 341-345Crossref PubMed Scopus (261) Google Scholar, 4Rao J.K. Bujacz G. Wlodawer A. FEBS Lett. 1998; 439: 133-137Crossref PubMed Scopus (140) Google Scholar, 5Eder M. Schlattner U. Becker A. Wallimann T. Kabsch W. Fritz-Wolf K. Prot. Sci. 1999; 8: 2258-2269Crossref PubMed Scopus (97) Google Scholar, 6Eder M. Fritz-Wolf K. Kabsch W. Wallimann T. Schlattner U. Proteins. 1999; 39: 216-225Crossref Google Scholar) and residues in the active site are highly conserved M. Schlattner U. Becker A. Wallimann T. Kabsch W. Fritz-Wolf K. Prot. Sci. 1999; 8: 2258-2269Crossref PubMed Scopus (97) Google Scholar, G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8449-8454Crossref PubMed Scopus (234) Google Scholar, M. T. Wallimann T. Wyss M. Mol. Cell. Biochem. 1994; Scopus Google Scholar), is that all guanidino a similar reaction mechanism. The AK transition state structure that the and have with substrates provide hydrogen that to substrate binding and is to two which are of the of the is with a and the of the and γ-phosphate of However, hydrogen be the only function of in CK, because with the also able to the hydrogen in a at of the domain by the binding of nucleotide M. M. P. Wallimann T. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), the negatively charged with The AK of a hydrogen at the active site which the negatively charged cluster to a containing the of cysteine In this cysteine with the and is not only for substrate binding also catalytic activity by the group and a partial positive charge away from the reactive (7Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8449-8454Crossref PubMed Scopus (234) Google Scholar). may the to because the amino acid at this is to hydrogen and may a critical of the enzyme In D228N, of providing hydrogen a of as well as the in the at the active could the lack of The residue in the negatively charged Glu227, is most in with the substrate The AK of Glu227 the group in an for away a partial positive charge from the substrate group to increase the of the to the guanidino group could also for the of octameric Glu227 to dissociate into alignment of is to be for the that to two glutamates were proposed as candidates for acid base including the of Glu227 (7Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8449-8454Crossref PubMed Scopus (234) Google Scholar). The of Glu227 in CK are with a function. Ser280 is by numbering) in the transition state structure of AK However, this residue is not to an as By contrast, is in hydrogen to the AK of which in is away from the substrate (7Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8449-8454Crossref PubMed Scopus (234) Google Scholar). In Ser280 may also the of the group of is with the low K m of S280D for because the charge of at this may the charged group However, has to also the structure of S280D as with near CD and The K m of S280A for different substrates is not from the AK we have identified several key residues in the active site of CK. on of proteins and the recently transition state structure of AK (7Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8449-8454Crossref PubMed Scopus (234) Google Scholar), we could also the putative of amino clearly that even residues that are not in with the substrates can to catalysis. CK a of with several residues involved a catalytic and charge at the active site may be to the high of guanidino alignment of both substrates and an for the transphosphorylation The AK transition state structure (7Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8449-8454Crossref PubMed Scopus (234) Google Scholar) a catalytic mechanism of guanidino including of of the substrates M.I. Proc. Natl. Acad. Sci. U. S. A. 1971; PubMed Scopus Google Scholar), Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. PubMed Google Scholar), the transition partial charge and also acid base catalysis. the latter on the AK structure (7Zhou G. Somasundaram T. Blanc E. Parthasarathy G. Ellington W.R. Chapman M.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8449-8454Crossref PubMed Scopus (234) Google Scholar), our data Glu227 as the most structural and be to the catalytic mechanism of guanidino at a molecular W. Kabsch and all of the Wallimann D. for and as well as A. W. and Z. for providing human sMtCK