Belogurov G A, Fabrichniy I P, Pohjanjoki P, Kasho V N, Lehtihuhta E, Turkina M V, Cooperman B S, Goldman A, Baykov A A, Lahti R
A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow 119899, Russia.
Biochemistry. 2000 Nov 14;39(45):13931-8. doi: 10.1021/bi000895s.
Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4. 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4. 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3). These data confirmed that enzyme-PP(i) intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP(i) release. The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PP(i) intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PP(i) intermediate decelerates PP(i) hydrolysis but accelerates PP(i) release by affecting the back isomerization. The binding of two Mg(2+) ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3. An acidic group with a pK(a) approximately 9 is modulatory in PP(i) hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.
在较宽的pH范围内测定了酵母无机焦磷酸酶的五种催化功能：稳态焦磷酸（PP(i)）水解（pH 4.8 - 10）和合成（pH 6.3 - 9.3）、磷酸 - 水氧交换（pH 4.8 - 9.3）、酶结合PP(i)的平衡形成（pH 4.8 - 9.3）以及镁离子（Mg(2+)）结合（pH 5.5 - 9.3）。这些数据证实了酶 - PP(i)中间体在反应循环中会发生异构化，并使得能够估算化学键断裂的微观速率常数以及PP(i)释放的宏观速率常数。研究发现，异构化作用会使酶 - PP(i)中间体中关键基团（可能是亲核水）的pK(a)从大于7降至5.85。异构化后的酶 - PP(i)中间体的质子化会减缓PP(i)水解，但通过影响逆向异构化来加速PP(i)释放。两个Mg(2+)离子与游离酶的结合需要大约五个平均pK(a)为6.3的碱性基团。一个pK(a)约为9的酸性基团在PP(i)水解和金属离子结合过程中起调节作用，这表明该基团维持着酶的整体结构，而非直接参与催化作用。
