Lee C S, O'Sullivan W J
J Biol Chem. 1985 Nov 15;260(26):13909-15.
The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) could act as substrates for phosphomevalonate kinase in the presence of Mg2+ and Cd2+ as activating divalent metal cations. The Sp diastereomer of ATP alpha S was the preferred substrate regardless of the metal ion used, consistent with the metal ion not binding to the alpha-phosphate. With ATP beta S, the Sp diastereomer was the preferred substrate with Mg2+, and the Rp diastereomer was the preferred substrate with Cd2+. The reversal of specificity establishes that the metal is chelated through the beta-phosphate in the active site of the phosphomevalonate kinase reaction. A comparison of the Vmax values as a function of substitution of oxygen by sulfur showed the order for Mg2+ to be: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Sp) greater than ATP gamma S greater than ATP beta S(Rp). With Cd2+ as the activating metal ion, the order was: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Rp) greater than ATP gamma S greater than ATP beta S(Sp). It is concluded that the chelate structure of metal ATP substrate in the phosphomevalonate kinase reaction is the delta, beta, gamma-bidentate complex. 31P NMR measurements and radioassay with [2-14C] phosphomevalonate were used to measure the equilibrium of the reaction catalyzed by phosphomevalonate kinase with ATP and phosphorothioate analogues of ATP as the phosphoryl group donor. The order as a phosphate donor as determined by both methods in the phosphomevalonate kinase reaction is ATP beta S greater than ATP alpha S greater than ATP greater than ATP gamma S. Except for ATP gamma S, the equilibrium is shifted in the direction of formation of ADP alpha S and ADP beta S relative to ADP formation. Thus, ATP beta S rather than ATP would be effective for the synthesis of diphosphomevalonate. The phosphomevalonate kinase reaction could also be used to synthesize mevalonate 5-(2-thiodiphosphate) using ATP gamma S as the phosphoryl group donor.
腺苷5'-O-(1-硫代三磷酸)(ATPαS)、腺苷5'-O-(2-硫代三磷酸)(ATPβS)和腺苷5'-O-(3-硫代三磷酸)(ATPγS)的非对映异构体在Mg2+和Cd2+作为激活二价金属阳离子存在的情况下可作为磷酸甲羟戊酸激酶的底物。无论使用何种金属离子，ATPαS的Sp非对映异构体都是首选底物，这与金属离子不与α-磷酸结合一致。对于ATPβS，Sp非对映异构体是Mg2+存在时的首选底物，而Rp非对映异构体是Cd2+存在时的首选底物。特异性的反转表明金属在磷酸甲羟戊酸激酶反应的活性位点通过β-磷酸螯合。比较作为氧被硫取代的函数的Vmax值，Mg2+的顺序为：ATP大于ATPαS(Sp)大于ATPαS(Rp)大于ATPβS(Sp)大于ATPγS大于ATPβS(Rp)。以Cd2+作为激活金属离子时，顺序为：ATP大于ATPαS(Sp)大于ATPαS(Rp)大于ATPβS(Rp)大于ATPγS大于ATPβS(Sp)。得出结论，磷酸甲羟戊酸激酶反应中金属ATP底物的螯合结构是δ,β,γ-双齿络合物。使用31P NMR测量和用[2-14C]磷酸甲羟戊酸进行放射性测定来测量磷酸甲羟戊酸激酶催化的以ATP和ATP的硫代磷酸酯类似物作为磷酰基供体的反应的平衡。在磷酸甲羟戊酸激酶反应中，两种方法确定的作为磷酸供体的顺序是ATPβS大于ATPαS大于ATP大于ATPγS。除了ATPγS，相对于ADP的形成，平衡向ADPαS和ADPβS形成的方向移动。因此，ATPβS而非ATP对于合成二磷酸甲羟戊酸将是有效的。磷酸甲羟戊酸激酶反应也可用于以ATPγS作为磷酰基供体合成甲羟戊酸5-(2-硫代二磷酸)。
