Ibrahim M M, Weber I T, Knudsen T B
Jefferson Medical College, Department of Pathology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Biochem Biophys Res Commun. 1995 Apr 17;209(2):407-16. doi: 10.1006/bbrc.1995.1518.
Human adenosine deaminase (ADA) cDNA was randomly mutagenized in vitro and bacterial transformants were selected for resistance to the potent enzyme inhibitor, pentostatin (dCF). Cells transformed with mutant plasmids dCF-R2 and dCF-R6 were able to grow in the presence of 10(-6) M dCF, whereas 10(-11) M dCF blocked growth of cells complemented with wild-type ADA. DNA sequence analysis revealed double G/A conversions mutating Lys11 and Gln255 in dCF-R2, and Gly 31 and Glu 99 in dCF-R6, to different amino acids. Located far from the enzyme active site, these substitutions did not greatly affect the Km or Ki of the enzyme but were predicted to destabilize interactions within the enzyme structure. Mutants such as these may be useful to analysis of the stereology of inhibitor binding to ADA and toxicity of dCF.
人腺苷脱氨酶（ADA）cDNA在体外进行随机诱变，然后选择对强效酶抑制剂喷司他丁（2'-脱氧助间型霉素，dCF）具有抗性的细菌转化体。用突变质粒dCF-R2和dCF-R6转化的细胞能够在10^(-6) M dCF存在的情况下生长，而10^(-11) M dCF则会抑制用野生型ADA互补的细胞生长。DNA序列分析显示，dCF-R2中赖氨酸11和谷氨酰胺255以及dCF-R6中甘氨酸31和谷氨酸99发生了双G/A转换，突变为不同的氨基酸。这些取代位于远离酶活性位点的位置，对酶的Km或Ki影响不大，但预计会破坏酶结构内的相互作用。这类突变体可能有助于分析抑制剂与ADA结合的立体化学以及dCF的毒性。
