Stevens L A, Moss J, Vaughan M, Pizza M, Rappuoli R
Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA.
Infect Immun. 1999 Jan;67(1):259-65. doi: 10.1128/IAI.67.1.259-265.1999.
Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsalpha, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction.
大肠杆菌热不稳定肠毒素（LT）是一种由一个A亚基（LTA）和五个B亚基组成的寡聚蛋白，它通过对Gsα进行ADP核糖基化发挥作用，Gsα是一种激活腺苷酸环化酶的鸟嘌呤核苷酸结合（G）蛋白。LTA还能对简单胍基化合物（如精氨酸）进行ADP核糖基化，并催化自身的自动ADP核糖基化。所有LTA催化的反应都被ADP核糖基化因子（ARFs）增强，ARFs是20 kDa的鸟嘌呤核苷酸结合蛋白。LTA中精氨酸-7（R7K）、缬氨酸-53（V53D）、丝氨酸-63（S63K）、缬氨酸97（V97K）或酪氨酸-104（Y104K）的替换导致完全组装但无毒的蛋白质。S63K、V53D和R7K是催化位点突变，而V97K和Y104K分别是催化位点相邻和外部的氨基酸替换。通过测量ADP核糖基转移酶活性（即自动ADP核糖基化和ADP核糖基胍丁胺合成）以及与ARF的相互作用（即抑制ARF刺激的霍乱毒素ADP核糖基转移酶活性和ARF对突变体自动ADP核糖基化的影响）来量化诱变的效果。所有突变体在ADP核糖基转移酶测定中均无活性；然而，在重组人ARF6存在的情况下检测到了自动ADP核糖基化，尽管比天然LT少得多（Y104K > V53D > V97K > R7K，S63K）。基于游离ADP核糖缺乏抑制作用，观察到的自动ADP核糖基化活性是酶促的，而不是由于游离ADP核糖的非酶促添加。V53D、S63K和R7K在阻断ARF对霍乱毒素ADP核糖基转移酶刺激方面比Y104K或V97K更有效。基于这些数据，似乎ARF结合位点和催化位点并不相同，并且NAD裂隙外部的一个区域可能参与LTA-ARF相互作用。
