Wise J G, Duncan T M, Latchney L R, Cox D N, Senior A E
Biochem J. 1983 Nov 1;215(2):343-50. doi: 10.1042/bj2150343.
Properties of purified F1-ATPase from Escherichia coli mutant strain AN484 (uncD412) have been studied in an attempt to understand why the amino acid substitution in the beta-subunit of this enzyme causes a tenfold reduction from normal MgATP hydrolysis rate. In most properties that were studied, uncD412 F1-ATPase resembled normal E. coli F1-ATPase. Both enzymes were found to contain a total of six adenine-nucleotide-binding sites, of which three were found to be non-exchangeable and three were exchangeable (catalytic) sites. Binding of the non-hydrolysable substrate analogue adenosine 5'-[beta gamma-imido]triphosphate (p[NH]ppA) to the three exchangeable sites showed apparent negative co-operativity. The binding affinities for p[NH]ppA, and also ADP, at the exchangeable sites were similar in the two enzymes. Both enzymes were inhibited by efrapeptin, aurovertin and p[NH]ppA, and were inactivated by dicyclohexylcarbodi-imide, 4-chloro-7-nitrobenzofurazan and p-fluorosulphonyl-benzoyl-5'-adenosine. Km values for CaATP and MgATP were similar in the two enzymes. uncD412 F1-ATPase was abnormally unstable at high pH, and dissociated into subunits readily with consequent loss of activity. The reason for the impairment of catalysis in uncD412 F1-ATPase cannot be stated with certainty from these studies. However we discuss the possibility that the mutation interrupts subunit interaction, thereby causing a partial impairment in the site-site co-operativity which is required for 'promotion' of catalysis in this enzyme.
为了弄清楚大肠杆菌突变株AN484（uncD412）中纯化的F1 - ATP酶的β亚基上的氨基酸取代为何会导致MgATP水解速率比正常情况降低十倍，人们对其性质进行了研究。在大多数所研究的性质方面，uncD412 F1 - ATP酶类似于正常的大肠杆菌F1 - ATP酶。两种酶都被发现总共含有六个腺嘌呤核苷酸结合位点，其中三个被发现是不可交换的，三个是可交换的（催化）位点。不可水解的底物类似物腺苷5'-[βγ - 亚氨基]三磷酸（p[NH]ppA）与三个可交换位点的结合表现出明显的负协同性。两种酶在可交换位点对p[NH]ppA以及ADP的结合亲和力相似。两种酶都受到依弗菌素、金轮菌素和p[NH]ppA的抑制，并被二环己基碳二亚胺、4 - 氯 - 7 - 硝基苯并呋喃和对氟磺酰苯甲酰 - 5'-腺苷灭活。两种酶对CaATP和MgATP的Km值相似。uncD412 F1 - ATP酶在高pH下异常不稳定，容易解离成亚基，从而导致活性丧失。从这些研究中无法确切说明uncD412 F1 - ATP酶催化受损的原因。然而，我们讨论了这种突变打断亚基相互作用的可能性，从而导致该酶催化“促进”所需的位点 - 位点协同性部分受损。
