Shibaev V N, Eliseeva G I, Kochetkov N K
Biochim Biophys Acta. 1975 Sep 22;403(1):9-16. doi: 10.1016/0005-2744(75)90003-0.
The interaction of alpha-D-glucopyranosyl pyrophosphates of 5-X-uridines (X = CH3, NH2, CH3O, I, Br, Cl, OH) with uridine diphosphate glucose (UDPGlc) dehydrogenase (EC 1.1.1.22) from calf liver has been studied. All the derivatives investigated were able to serve as substrates for the enzyme. The apparent Michaelis constants for UDPGlc-analogs were dependent both on electronic and steric factors. Increase of substituent negative inductive effect lead to decrease of pKa for ionization of the NH-group in the uracil nucleus and, consequently, to a diminishing of the proportion of the active analog species under the conditions of assay. After correction for the ionization effect, the Km values were found to depend on the van der Waals radius of the substituent. The value of 1.95 A seems to be critical, as the analogs with bulkier substituents at C-5 showed a decreased affinity to the enzyme. The maximal velocity values of the analogs were also dependent on nature of the substituent. Good linear correlation between log V and substituent hydrophobic phi-constant was observed for a number of the analogs, although V values for the nucleotides with X = H, OH or NH2 were higher than would be expected on the basis of the correlation. The significance of the results for understanding of the topography of UDPGlc dehydrogenase active site is discussed.
对5-X-尿苷（X = CH3、NH2、CH3O、I、Br、Cl、OH）的α-D-吡喃葡萄糖基焦磷酸酯与来自小牛肝脏的尿苷二磷酸葡萄糖（UDPGlc）脱氢酶（EC 1.1.1.22）之间的相互作用进行了研究。所有研究的衍生物都能够作为该酶的底物。UDPGlc类似物的表观米氏常数取决于电子和空间因素。取代基负诱导效应的增加导致尿嘧啶核中NH基团电离的pKa降低，因此，在测定条件下活性类似物物种的比例降低。校正电离效应后，发现Km值取决于取代基的范德华半径。1.95 Å的值似乎很关键，因为在C-5处具有更大取代基的类似物对该酶的亲和力降低。类似物的最大速度值也取决于取代基的性质。对于许多类似物，观察到log V与取代基疏水φ常数之间具有良好的线性相关性，尽管X = H、OH或NH2的核苷酸的V值高于基于该相关性预期的值。讨论了这些结果对于理解UDPGlc脱氢酶活性位点拓扑结构的意义。
