Ghosh M C, Jencks W P
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
Biochemistry. 1996 Sep 24;35(38):12587-90. doi: 10.1021/bi961324w.
Phosphorylation of the free sodium-potassium adenosinetriphosphatase of sheep kidney upon the addition of 0.02-2.0 mM ATP and a saturating concentration of Na+ (125 mM) follows pseudo-first-order kinetics. The first-order rate constant increases with increasing [ATP] and levels off at high [ATP] with a limiting rate constant of 180 s-1 at 25 degrees C, pH 7.4, 125 mM NaCl, and 3.0 mM MgCl2. This rate constant is about 1/3 of the maximum rate constant of 460 s-1 for phosphorylation of enzyme that had been preincubated with Na+ under identical conditions [Keillor, J. W., & Jencks, W. P., (1996) Biochemistry 35, 2750-2753]; this rate ratio is similar to that for phosphorylation of the calcium ATPase with and without initial incubation with Ca2+ [Petithory, J. R., & Jencks, W. P. (1986) Biochemistry 25, 4493-4497]. The K0.5 for ATP is 18 +/- 3 microM for the free enzyme, which is about 1/4 of K0.5 = 75 microM for enzyme that was preincubated with Na+. Addition of ADP to ATP and Na+ decreases the yield of E approximately P progressively with increasing ADP concentration. Upon an increase of [ADP] from 0 to 2.0 mM, the rate constant for phosphorylation decreases 4-fold (167 to 41 s-1) at low [ATP] (0.25 mM) and about 2.7-fold (174 to 64 s-1) at high [ATP] (2.0 mM). The absence of an induction period for phosphorylation of E upon the addition of saturating concentrations of ATP and Na+ indicates that all the prior reaction steps are much faster than the rate-limiting step. These results are consistent with a rate-determining conformational change of the E.ATP.Na3 intermediate. The decrease of the rate constant with increasing [ADP] is attributed to competition between ATP and ADP for the free enzyme.
在添加0.02 - 2.0 mM ATP和饱和浓度的Na⁺（125 mM）后，绵羊肾游离钠钾三磷酸腺苷酶的磷酸化遵循假一级动力学。在25℃、pH 7.4、125 mM NaCl和3.0 mM MgCl₂条件下，一级速率常数随[ATP]增加而增大，并在高[ATP]时趋于平稳，极限速率常数为180 s⁻¹。该速率常数约为在相同条件下与Na⁺预孵育的酶磷酸化最大速率常数460 s⁻¹的1/3 [Keillor, J. W., & Jencks, W. P., (1996) Biochemistry 35, 2750 - 2753]；该速率比类似于钙ATP酶在有无初始Ca²⁺孵育情况下磷酸化的速率比 [Petithory, J. R., & Jencks, W. P. (1986) Biochemistry 25, 4493 - 4497]。游离酶的ATP的K₀.₅为18 ± 3 μM，约为与Na⁺预孵育的酶的K₀.₅ = 75 μM的1/4。向ATP和Na⁺中添加ADP会随着ADP浓度增加而逐渐降低E≈P的产率。当[ADP]从0增加到2.0 mM时，在低[ATP]（0.25 mM）下磷酸化的速率常数降低4倍（从167降至41 s⁻¹），在高[ATP]（2.0 mM）下约降低2.7倍（从174降至64 s⁻¹）。添加饱和浓度的ATP和Na⁺后E磷酸化不存在诱导期，这表明所有先前的反应步骤都比限速步骤快得多。这些结果与E.ATP.Na₃中间体的限速构象变化一致。速率常数随[ADP]增加而降低归因于ATP和ADP对游离酶的竞争。
