Determination of the affinity of each component of a composite quaternary transition-state analogue complex of creatine kinase

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 degreesC 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 muM, 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 Kd for M-ADP remains essentially unchanged at 132 +/- 10 muM, while Delta%F decreases slightly to 13.2 +/- 0.3%. In the presence of 10 mM nitrate, but no creatine, the apparent Kd is once again essentially unchanged at 143 23 muM, 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 Kd for M-ADP 10-fold to 13.7 +/- 0.7 mM, and Delta%F increases to 20.6 0.3%, strongly suggesting that the simultaneous presence of saturating levels ofc-reatine and nitrate increases the affinity of CK for MgADP and promotes the formation of the enzyme(.)M(.)ADPZ 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 Kd values for MgADP of 132 +/- 10 muM, 25.2 +/- 1.3 muM, 18.8 +/- 0.9 muM, 13.7 +/- 0.7 muM, and 6.4 +/- 0.7 muM 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 trim creatine and various fixed concentrations of NaNO3, the apparent Kd for MgADP decreases with increasing fixed concentrations of nitrate. A plot of the apparent Kd for MgADP vs [NO3-] suggests a Kd 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) M-3. To our knowledge this is the first reported dissociation constant for a ternary or quaternary TSAC.