Magnesium−Adenosine Diphosphate Binding Sites in Wild-type Creatine Kinase and in Mutants:  Role of Aromatic Residues Probed by Raman and Infrared Spectroscopies

Hagemann, Hans‐Rudolf; Marcillat, Olivier; Buchet, René; Vial, Christian

doi:10.1021/bi000009d

Cited by 6 publications

(4 citation statements)

References 55 publications

(76 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, despite these many investigations, the precise catalytic role of the cysteine remains to be determined. More readily interpreted results confirm that both arginine () and tryptophan ( − ) residues function as nucleotide-binding groups. Finally, mutagenesis of all five highly conserved histidine residues have excluded a centrally important catalytic function for any of them ().…”

mentioning

confidence: 58%

Mutagenesis of Two Acidic Active Site Residues in Human Muscle Creatine Kinase: Implications for the Catalytic Mechanism

et al. 2001

View full text Add to dashboard Cite

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.

show abstract

mentioning

confidence: 58%

Mutagenesis of Two Acidic Active Site Residues in Human Muscle Creatine Kinase: Implications for the Catalytic Mechanism

et al. 2001

View full text Add to dashboard Cite

show abstract

“…Escherichia coli BL21 (DE3) cells were transformed with the recombinant plasmid pET21-MCK [38], this plasmid drives the expression of a full-length muscle CK without any purification tag. For NMR experiments, we prepared different sets of isotopically labelled MCK samples.…”

Section: Expression Of Mckmentioning

confidence: 99%

Dynamical properties of the loop 320s of substrate‐free and substrate‐bound muscle creatine kinase by NMR

Rivière¹,

Hologne²,

Marcillat³

et al. 2012

The FEBS Journal

Self Cite

View full text Add to dashboard Cite

Muscle creatine kinase (MCK; EC 2.7.3.2) is a 86 kDa homodimer that belongs to the family of guanidino kinases. MCK has been intensively studied for several decades, but it is still not known why it is a dimer because this quaternary structure does not translate into obvious structural or functional advantages over the homologous monomeric arginine kinase. In particular, it remains to be demonstrated whether MCK subunits are independent. Here, we describe NMR chemical-shift perturbation and relaxation experiments designed to study the active site 320s flexible loop of this enzyme. The analysis was performed with the enzyme in its ligandfree and MgADP-complexed forms, as well as with the transition-state analogue abortive complex (MCK-Mg-ADP-creatine-nitrate ion). Our data indicate that each subunit can bind substrates independently.

show abstract

“…In the first three decades, structure−function information about CK was obtained by enzyme kinetic, chemical modification, and spectroscopic studies (see refs 1 and 2 for reviews). The past 15 years have seen new approaches added to the arsenal for the attack on the structure−function relationships in CK and other guanidino kinases, including site-directed mutagenesis ( − ) and X-ray crystallography ( − ).…”

mentioning

confidence: 99%

Determination of the Affinity of Each Component of a Composite Quaternary Transition-State Analogue Complex of Creatine Kinase

et al. 2002

View full text Add to dashboard Cite

Recombinant rabbit muscle creatine kinase (CK) was titrated with MgADP in 50 mM Bicine and 5 mM Mg(OAc)2, pH 8.3, at 30.0 degrees C by following a decrease in the protein's intrinsic fluorescence. In the presence of 50 mM NaOAc, but in the absence of added creatine or nitrate, MgADP has an apparent K(d) of 135 +/- 7 microM, and the total change in fluorescence on saturation (Delta%F) is 15.3 +/- 0.3%. Acetate was used as the anion in this experiment because it does not promote the formation of a CK.MgADP.anion.creatine transition-state analogue complex (TSAC) [Millner-White and Watts (1971) Biochem. J. 122, 727-740]. In the presence of 80 mM creatine, but no nitrate, the apparent K(d) for MgADP remains essentially unchanged at 132 +/- 10 microM, while Delta%F decreases slightly to 13.2 +/- 0.3%. In the presence of 10 mM nitrate, but no creatine, the apparent K(d) is once again essentially unchanged at 143 +/- 23 microM, but the Delta%F is markedly reduced to 4.2 +/- 0.2%. The presence of both 10 mM nitrate and 80 mM creatine during titration reduces the apparent K(d) for MgADP 10-fold to 13.7 +/- 0.7 microM, and Delta%F increases to 20.6 +/- 0.3%, strongly suggesting that the simultaneous presence of saturating levels of creatine and nitrate increases the affinity of CK for MgADP and promotes the formation of the enzyme*MgADP*nitrate*creatine TSAC. When the fluorescence of CK was titrated with MgADP in the presence of 80 mM creatine and fixed saturating concentrations of various anions, apparent K(d) values for MgADP of 132 +/- 10 microM, 25.2 +/- 1.3 microM, 18.8 +/- 0.9 microM, 13.7 +/- 0.7 microM, and 6.4 +/- 0.7 microM were observed as the anion was changed from acetate to formate to chloride to nitrate to nitrite, respectively. This is the same trend reported by Millner-White and Watts for the effectiveness of various monovalent anions in forming the CK.MgADP.anion.creatine TSAC. On titration of CK with MgADP in the presence of 80 mM creatine and various fixed concentrations of NaNO3, the apparent K(d) for MgADP decreases with increasing fixed concentrations of nitrate. A plot of the apparent K(d) for MgADP vs [NO3-] suggests a K(d) for nitrate from the TSAC of 0.39 +/- 0.07 mM. Similarly, titration with MgADP in the presence of 10 mM NaNO3 and various fixed concentrations of creatine gives a value of 0.9 +/- 0.4 mM for the dissociation of creatine from the TSAC. The data were used to calculate K(TDAC), the dissociation constant of the quaternary TSAC into its individual components, of 3 x 10(-10) M3. To our knowledge this is the first reported dissociation constant for a ternary or quaternary TSAC.

show abstract

Magnesium−Adenosine Diphosphate Binding Sites in Wild-type Creatine Kinase and in Mutants: Role of Aromatic Residues Probed by Raman and Infrared Spectroscopies

Cited by 6 publications

References 55 publications

Mutagenesis of Two Acidic Active Site Residues in Human Muscle Creatine Kinase: Implications for the Catalytic Mechanism

Mutagenesis of Two Acidic Active Site Residues in Human Muscle Creatine Kinase: Implications for the Catalytic Mechanism

Dynamical properties of the loop 320s of substrate‐free and substrate‐bound muscle creatine kinase by NMR

Determination of the Affinity of Each Component of a Composite Quaternary Transition-State Analogue Complex of Creatine Kinase

Contact Info

Product

Resources

About