tailieunhanh - Báo cáo khoa học: Binding of non-natural 3¢-nucleotides to ribonuclease A

2¢-Fluoro-2¢-deoxyuridine 3¢-phosphate (dU F MP) and arabinouridine 3¢-phosphate (araUMP) have non-natural furanose rings. dU F MP and araUMP were prepared by chemical synthesis and found to have three- to sevenfold higher affinity than uridine 3¢-phosphate (3¢-UMP) or 2¢-deoxy-uridine 3¢-phosphate (dUMP) for ribonuclease A (RNase A). These differ-ences probably arise (in part) from the phosphoryl groups of 3¢-UMP, dU F MP, and araUMP (pKa¼) being more anionic than that of dUMP (pKa¼). . | iFEBS Journal Binding of non-natural 3 -nucleotides to ribonuclease A Cara L. Jenkins1 Nethaji Thiyagarajan2 Rozamond Y. Sweeney3 Michael P. Guy3 Bradley R. Kelemen3 K. Ravi Acharya2 and Ronald T. Raines1 3 1 Department of Chemistry University of Wisconsin-Madison WI USA 2 Department of Biology and Biochemistry University of Bath UK 3 Department of Biochemistry University of Wisconsin-Madison WI USA Keywords arabinonucleotide enzyme inhibitor 2 -fluoro-2 -deoxynucleotide ribonuclease X-ray crystallography Correspondence K. R. Acharya Department of Biology and Biochemistry University of Bath Claverton Down Bath BA2 7AY UK Fax 44 1225 386779 Tel. 44 1225 386238 E-mail R. T. Raines Department of Biochemistry University of Wisconsin-Madison 433 Babcock Drive Madison WI 53706-1544 USA Fax 1 608 262 3453 Tel 1 608 262 8588 E-mail raines@ Received 4 October 2004 revised 24 November 2004 accepted 2 December 2004 2 -Fluoro-2 -deoxyuridine 3 -phosphate dUFMP and arabinouridine 3 -phosphate araUMP have non-natural furanose rings. dUFMP and araUMP were prepared by chemical synthesis and found to have three- to sevenfold higher affinity than uridine 3 -phosphate 3 -UMP or 2 -deoxy-uridine 3 -phosphate dUMP for ribonuclease A RNase A . These differences probably arise in part from the phosphoryl groups of 3 -UMP dUFMP and araUMP pKa being more anionic than that of dUMP pKa . The three-dimensional structures of the crystalline complexes of RNase A with dUMP dUFMP and araUMP were determined at A resolution by X-ray diffraction analysis. In these three structures the uracil nucleobases and phosphoryl groups bind to the enzyme in a nearly identical position. Unlike 3 -UMP and dUFMP dUMP and ara-UMP bind with their furanose rings in the preferred pucker. In the RNase A-araUMP complex the 2 -hydroxyl group is exposed to the solvent. All four 3 -nucleotides bind more tightly to wild-type RNase A than to its T45G variant which lacks the