Pieper U, Brinkmann T, Krüger T, Noyer-Weidner M, Pingoud A
Institut für Biochemie (FB 15), Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, Giessen, D-35392, Germany.
J Mol Biol. 1997 Sep 19;272(2):190-9. doi: 10.1006/jmbi.1997.1228.
McrBC, a GTP-dependent restriction enzyme from E. coli K-12, cleaves DNA containing methylated cytosine residues 40 to 80 residues apart and 3'-adjacent to a purine residue (PumCN40-80PumC). The presence of the three consensus sequences characteristic for guanine nucleotide binding proteins in one of the two subunits of McrBC suggests that this subunit is responsible for GTP binding and hydrolysis. We show here that (i) McrB binds GTP with an affinity of 10(6) M-1 and that GTP binding stabilizes McrB against thermal denaturation. (ii) McrB binds GDP about 50-fold and ATP at least three orders of magnitude more weakly than GTP. (iii) McrB hydrolyzes GTP in the presence of Mg2+ with a steady-state rate of approximately 0.5 min-1. (iv) McrC stimulates GTP hydrolysis 30-fold, but substrate DNA has no detectable effect on the GTPase activity of McrB, neither by itself nor in the presence of McrC. (v) Substitution of N339 and N376 with alanine allowed us to identify NTAD (339 to 342) rather than NKKA (376 to 379) as the equivalent of the third consensus sequence motif characteristic for guanine nucleotide binding proteins, NKXD.
McrBC是一种来自大肠杆菌K-12的依赖GTP的限制酶，可切割含有甲基化胞嘧啶残基的DNA，这些残基相隔40至80个残基，且位于嘌呤残基（PumCN40 - 80PumC）的3'端相邻位置。McrBC两个亚基之一中存在鸟嘌呤核苷酸结合蛋白特有的三个共有序列，这表明该亚基负责GTP的结合和水解。我们在此表明：（i）McrB以10⁶ M⁻¹的亲和力结合GTP，且GTP结合使McrB对热变性稳定。（ii）McrB结合GDP的亲和力约为结合GTP的50倍，结合ATP的亲和力比结合GTP至少弱三个数量级。（iii）McrB在Mg²⁺存在下以约0.5 min⁻¹的稳态速率水解GTP。（iv）McrC将GTP水解刺激30倍，但底物DNA对McrB的GTP酶活性没有可检测到的影响，无论是其单独存在还是在McrC存在的情况下。（v）用丙氨酸取代N339和N376使我们能够确定NTAD（339至342）而非NKKA（376至379）等同于鸟嘌呤核苷酸结合蛋白特有的第三个共有序列基序NKXD。
