Mizuguchi H, Cook P F, Hasemann C A, Uyeda K
Research Service, Department of Veterans' Affairs Medical Center, Dallas, Texas 75216, USA.
Biochemistry. 1997 Jul 22;36(29):8775-84. doi: 10.1021/bi970639o.
A bifunctional enzyme, fructose-6-phosphate 2-kinase-fructose 2, 6-bisphosphatase, catalyzes synthesis and degradation of fructose 2, 6-bisphosphate. Mutants of basic residues, including Lys51, Arg78, Arg79, Arg136, Lys172, and Arg193, immediately around the active site of rat testis fructose 6-P,2-kinase were constructed, and their steady state kinetics, ATP binding, and the effect of pH on the kinetics were characterized. All mutants showed a several-fold increase in KMgATP, much larger increases in KFru 6-P, and decreased V compared to those of the wild type enzyme (WT). Replacement of Lys172 and Arg193 with Ala and Leu, respectively, also produced mutants with large KFru 6-P values. Substitution of Lys51, which is located in a Walker-A motif (GXXGXGKT, amino acids 45-52), with Ala or His resulted in enzymes with increased KMgATP values and unable to bind Fru 6-P. The dissociation constants for 2'(3')-O-(N-methylanthraniloyl)-ATP (mantATP) and ATP of all these mutants except Lys51 were similar. Lys51 mutants were unable to bind mantATP. The pH dependence of V and the V/Ks for MgATP and Fru 6-P suggest a mechanism in which reactants and enzyme combine irrespective of the protonation state of groups required for binding and catalysis, but only the correctly protonated enzyme-substrate complex is catalytically active. A chemical mechanism is suggested in which a general base accepts a proton from the 2-hydroxyl of Fru 6-P concomitant with nucleophilic attack on the gamma-phosphate of MgATP. Phosphoryl transfer is also facilitated by interaction of the gamma-phosphate with a positively charged residue that neutralizes the remaining negative charge. The dianionic form of the 6-phosphate of fructose 6-P is required for binding, and it is likely anchored by a positively charged enzyme residue. A comparison of the pH dependence of kinetic parameters for Ala or His mutant proteins at Lys51, Lys172, and Arg79 suggests that Lys51 interacts with the gamma-phosphate of MgATP and that several other arginines likely participate in transition state stabilization of the transferred phosphoryl. The active site general base has yet to be identified.
一种双功能酶，即6-磷酸果糖-2-激酶-果糖-2,6-二磷酸酶，催化果糖-2,6-二磷酸的合成与降解。构建了大鼠睾丸6-磷酸果糖激酶活性位点周围包括赖氨酸51、精氨酸78、精氨酸79、精氨酸136、赖氨酸172和精氨酸193等碱性残基的突变体，并对其稳态动力学、ATP结合以及pH对动力学的影响进行了表征。与野生型酶（WT）相比，所有突变体的KMgATP均增加了几倍，KFru 6-P增加得更多，而V降低。分别用丙氨酸和亮氨酸取代赖氨酸172和精氨酸193，也产生了KFru 6-P值较大的突变体。位于沃克A基序（GXXGXGKT，氨基酸45-52）中的赖氨酸51被丙氨酸或组氨酸取代后，产生的酶KMgATP值增加且无法结合6-磷酸果糖。除赖氨酸51突变体外，所有这些突变体的2'(3')-O-(N-甲基邻氨基苯甲酰基)-ATP（mantATP）和ATP的解离常数相似。赖氨酸51突变体无法结合mantATP。V以及MgATP和6-磷酸果糖的V/Ks对pH的依赖性表明，一种机制是反应物和酶的结合与结合和催化所需基团的质子化状态无关，但只有正确质子化的酶-底物复合物才具有催化活性。提出了一种化学机制，即一个通用碱从6-磷酸果糖的2-羟基接受一个质子，同时对MgATP的γ-磷酸进行亲核攻击。γ-磷酸与一个带正电荷的残基相互作用，中和剩余的负电荷，也促进了磷酰基转移。6-磷酸果糖的6-磷酸二阴离子形式是结合所必需的，并且可能由一个带正电荷的酶残基锚定。对赖氨酸51、赖氨酸172和精氨酸79处丙氨酸或组氨酸突变蛋白动力学参数的pH依赖性比较表明，赖氨酸51与MgATP的γ-磷酸相互作用，其他几个精氨酸可能参与转移磷酰基的过渡态稳定。活性位点通用碱尚未确定。
