Anashkin Viktor A, Salminen Anu, Tuominen Heidi K, Orlov Victor N, Lahti Reijo, Baykov Alexander A
From the Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland and the Belozersky Institute of Physico-Chemical Biology and Department of Chemistry, Lomonosov Moscow State University, Moscow 119899, Russia.
From the Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland and.
J Biol Chem. 2015 Nov 13;290(46):27594-603. doi: 10.1074/jbc.M115.680272. Epub 2015 Sep 23.
Among numerous proteins containing pairs of regulatory cystathionine β-synthase (CBS) domains, family II pyrophosphatases (CBS-PPases) are unique in that they generally contain an additional DRTGG domain between the CBS domains. Adenine nucleotides bind to the CBS domains in CBS-PPases in a positively cooperative manner, resulting in enzyme inhibition (AMP or ADP) or activation (ATP). Here we show that linear P(1),P(n)-diadenosine 5'-polyphosphates (ApnAs, where n is the number of phosphate residues) bind with nanomolar affinity to DRTGG domain-containing CBS-PPases of Desulfitobacterium hafniense, Clostridium novyi, and Clostridium perfringens and increase their activity up to 30-, 5-, and 7-fold, respectively. Ap4A, Ap5A, and Ap6A bound noncooperatively and with similarly high affinities to CBS-PPases, whereas Ap3A bound in a positively cooperative manner and with lower affinity, like mononucleotides. All ApnAs abolished kinetic cooperativity (non-Michaelian behavior) of CBS-PPases. The enthalpy change and binding stoichiometry, as determined by isothermal calorimetry, were ~10 kcal/mol nucleotide and 1 mol/mol enzyme dimer for Ap4A and Ap5A but 5.5 kcal/mol and 2 mol/mol for Ap3A, AMP, ADP, and ATP, suggesting different binding modes for the two nucleotide groups. In contrast, Eggerthella lenta and Moorella thermoacetica CBS-PPases, which contain no DRTGG domain, were not affected by ApnAs and showed no enthalpy change, indicating the importance of the DTRGG domain for ApnA binding. These findings suggest that ApnAs can control CBS-PPase activity and hence affect pyrophosphate level and biosynthetic activity in bacteria.
在众多含有成对调节性胱硫醚β-合酶（CBS）结构域的蛋白质中，II型焦磷酸酶（CBS-PPases）很独特，因为它们通常在CBS结构域之间还含有一个额外的DRTGG结构域。腺嘌呤核苷酸以正协同方式与CBS-PPases中的CBS结构域结合，导致酶抑制（AMP或ADP）或激活（ATP）。在此我们表明，线性P(1),P(n)-二腺苷5'-多磷酸（ApnAs，其中n是磷酸残基的数量）以纳摩尔亲和力与含DRTGG结构域的哈氏脱硫肠状菌、诺维梭菌和产气荚膜梭菌的CBS-PPases结合，并分别将它们的活性提高30倍、5倍和7倍。Ap4A、Ap5A和Ap6A以非协同方式且以类似的高亲和力与CBS-PPases结合，而Ap3A以正协同方式且以较低亲和力结合，类似于单核苷酸。所有ApnAs都消除了CBS-PPases的动力学协同性（非米氏行为）。通过等温滴定量热法测定，Ap4A和Ap5A的焓变和结合化学计量分别为每摩尔核苷酸约10千卡和每摩尔酶二聚体1摩尔，但Ap3A、AMP、ADP和ATP为5.5千卡/摩尔和2摩尔/摩尔，这表明这两类核苷酸基团的结合模式不同。相比之下，不含DRTGG结构域的迟缓埃格特菌和嗜热栖热菌的CBS-PPases不受ApnAs影响，且无焓变，这表明DTRGG结构域对ApnA结合很重要。这些发现表明，ApnAs可以控制CBS-PPase活性，从而影响细菌中的焦磷酸水平和生物合成活性。
