| Abstract
| - Recombinant rabbit muscle creatine kinase (CK) was titrated with MgADP in 50 mM Bicineand 5 mM Mg(OAc)2, pH 8.3, at 30.0 °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 apparentKd of 135 ± 7 μM, and the total change in fluorescence on saturation (Δ%F) is 15.3 ± 0.3%. Acetatewas 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 MgADP remainsessentially unchanged at 132 ± 10 μM, while Δ%F decreases slightly to 13.2 ± 0.3%. In the presenceof 10 mM nitrate, but no creatine, the apparent Kd is once again essentially unchanged at 143 ± 23 μM,but the Δ%F is markedly reduced to 4.2 ± 0.2%. The presence of both 10 mM nitrate and 80 mMcreatine during titration reduces the apparent Kd for MgADP 10-fold to 13.7 ± 0.7 μM, and Δ%F increasesto 20.6 ± 0.3%, strongly suggesting that the simultaneous presence of saturating levels of creatine andnitrate 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 mMcreatine and fixed saturating concentrations of various anions, apparent Kd values for MgADP of 132 ±10 μM, 25.2 ± 1.3 μM, 18.8 ± 0.9 μM, 13.7 ± 0.7 μM, and 6.4 ± 0.7 μM were observed as the anionwas changed from acetate to formate to chloride to nitrate to nitrite, respectively. This is the same trendreported by Millner-White and Watts for the effectiveness of various monovalent anions in forming theCK·MgADP·anion·creatine TSAC. On titration of CK with MgADP in the presence of 80 mM creatineand various fixed concentrations of NaNO3, the apparent Kd for MgADP decreases with increasing fixedconcentrations of nitrate. A plot of the apparent Kd for MgADP vs [NO3-] suggests a Kd for nitrate fromthe TSAC of 0.39 ± 0.07 mM. Similarly, titration with MgADP in the presence of 10 mM NaNO3 andvarious fixed concentrations of creatine gives a value of 0.9 ± 0.4 mM for the dissociation of creatinefrom the TSAC. The data were used to calculate KTDAC, the dissociation constant of the quaternary TSACinto its individual components, of 3 × 10-10 M3. To our knowledge this is the first reported dissociationconstant for a ternary or quaternary TSAC.
|
