Bullough D A, Verburg J G, Yoshida M, Allison W S
J Biol Chem. 1987 Aug 25;262(24):11675-83.
The characteristics of ATP hydrolysis at a single catalytic site of the bovine heart F1-ATPase (MF1) as originally described by Grubmeyer et al. (Grubmeyer, C., Cross, R.L., and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12092-12100) were compared with those of various chemically modified preparations of MF1 in which the steady state activity was severely attenuated. Although it was not necessary to age our preparations of native MF1 in the presence of 2 mM Pi to observe the same characteristics of single site catalysis, such aging did shift the equilibrium of bound substrate and bound products at the single catalytic site in favor of ATP. After loading a single catalytic site on the enzyme with substoichiometric [alpha,gamma-32P]ATP, the addition of 5-20 microM ATP or ADP was effective in promoting both the hydrolysis of bound [alpha,gamma-32P]ATP and release of radioactive products. Under these conditions, the 5-20 microM ATP added as promoter was hydrolyzed at a rate commensurate with the turnover rate of the enzyme, whereas the promoted hydrolysis of the [alpha,gamma-32P]ATP, preloaded at a single catalytic site, was considerably slower. Therefore, the high affinity, single catalytic site loaded first does not directly contribute to steady state ATP hydrolysis. That the single, high affinity catalytic site is not a "normal" catalytic site is supported by the properties of enzyme modified by 5'-p-fluorosulfonylbenzoyladenosine which exhibits only slightly altered characteristics of single site catalysis and promoted single site catalysis, despite exhibiting severely attenuated steady state turnover. Other modified forms of the enzyme in which the steady state activity was severely attenuated by derivatization with 5'-p-fluorosulfonylbenzoylinosine, 7-chloro-4-nitrobenzofurazan, or 1,5-difluoro-2,4-dinitrobenzene also bound substoichiometric ATP at a single catalytic site. However, the characteristics of single site hydrolysis by these modified forms of the enzyme differed considerably from those of native MF1.
将最初由格鲁布迈尔等人（格鲁布迈尔，C.，克罗斯，R.L.，和佩内夫斯基，H.S.（1982年）《生物化学杂志》257卷，12092 - 12100页）描述的牛心F1 - ATP酶（MF1）单个催化位点处ATP水解的特征，与稳态活性严重减弱的各种化学修饰的MF1制剂的特征进行了比较。尽管在2 mM磷酸根离子存在下对天然MF1制剂进行老化处理并非观察单个位点催化相同特征所必需，但这种老化确实使单个催化位点处结合底物和结合产物的平衡向有利于ATP的方向移动。在用亚化学计量的[α,γ - 32P]ATP加载酶上的单个催化位点后，添加5 - 20 μM ATP或ADP有效地促进了结合的[α,γ - 32P]ATP的水解以及放射性产物的释放。在这些条件下，作为促进剂添加的5 - 20 μM ATP以与酶的周转速率相当的速率水解，而在单个催化位点预先加载的[α,γ - 32P]ATP的促进水解则相当缓慢。因此，首先加载的高亲和力单个催化位点对稳态ATP水解没有直接贡献。5'-对氟磺酰苯甲酰腺苷修饰的酶的性质支持了单个高亲和力催化位点不是“正常”催化位点这一观点，尽管该酶的稳态周转严重减弱，但其单个位点催化和促进单个位点催化的特征仅略有改变。其他通过用5'-对氟磺酰苯甲酰肌苷、7 - 氯 - 4 - 硝基苯并呋喃或1,5 - 二氟 - 二硝基苯衍生化而使稳态活性严重减弱的酶的修饰形式，在单个催化位点也结合亚化学计量的ATP。然而，这些酶的修饰形式的单个位点水解特征与天然MF1的特征有很大不同。
