Kossykh V G, Schlagman S L, Hattman S
Department of Biology, University of Rochester, NY 14627, USA.
FEBS Lett. 1995 Aug 14;370(1-2):75-7. doi: 10.1016/0014-5793(95)00795-b.
ProCys in the conserved sequence motif IV of [cytosine-C5]-DNA methyltransferases is known to be part of the catalytic site. The Cys residue is directly involved in forming a covalent bond with the C6 of the target cytosine. We have found that substitution of Pro-185 with either Ala or Ser resulted in a reduced rate of methyl group transfer by the EcoRII DNA methyltransferase. In addition, we observed an increase in the Km for substrate S-adenosyl-L-methionine (AdoMet), but a decrease in the Km for substrate DNA. This is reflected in minor changes in kcat/Km for DNA, but in 10- to 100-fold reductions in kcat/Km for AdoMet. This suggests that Pro-185 is important to properly orient the activated cytosine and AdoMet for methyl group transfer by direct interaction with AdoMet and indirectly via the Cys interaction with cytosine.
已知[胞嘧啶 - C5] - DNA甲基转移酶保守序列基序IV中的脯氨酸 - 半胱氨酸（ProCys）是催化位点的一部分。半胱氨酸残基直接参与与目标胞嘧啶的C6形成共价键。我们发现，用丙氨酸或丝氨酸取代Pro - 185会导致EcoRII DNA甲基转移酶的甲基转移速率降低。此外，我们观察到底物S - 腺苷 - L - 甲硫氨酸（AdoMet）的米氏常数（Km）增加，但底物DNA的Km降低。这反映在DNA的催化常数与米氏常数之比（kcat/Km）有微小变化，但AdoMet的kcat/Km降低了10至100倍。这表明Pro - 185对于通过与AdoMet直接相互作用以及通过半胱氨酸与胞嘧啶的相互作用间接正确定向活化的胞嘧啶和AdoMet以进行甲基转移很重要。
