title: Identification of the active site nucleophile in nucleoside 2-deoxyribosyltransferase as glutamic acid 98.
abstract: 2'-Fluoro-2'-deoxyarabinonucleosides are time-dependent inhibitors of nucleoside 2-deoxyribosyltransferase. 2,6-Diamino-9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-9H-purine (dFDAP) inhibited the enzyme by formation of a primary complex (Kd = 140 microM) that isomerized to a secondary complex with a first-order rate constant of 0.2 min-1. Inhibited enzyme contained stoichiometric amounts of covalently bound 2'-fluoro-2'-deoxyarabinosyl moiety, recovered less than 5% of its activity after storage for a week at 5 degrees C, but regained over 70% of the lost activity by treatment with 600 microM Ade. 6-Amino-9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-9H-purine (dFAdo) was a product of the reactivation reaction. Proteolysis of inhibited enzyme identified a modified fragment that spanned residues 82-107 which could not be sequenced past Gly-96. dFDAP-inhibited enzyme and enzyme reacted with normal substrates (i.e. dThd and dAdo) were hydrolyzed between Met-97 and Glu-98 by 0.1 M NaOH. These findings and model studies on the base lability of peptides containing glutamyl esters suggested that the gamma-carboxylate of Glu-98 was esterfied during catalysis. The role of Glu-98 was confirmed by changing this residue to alanine. The specific activity of wild-type enzyme was 3 orders of magnitude greater than that of the mutant enzyme. Collectively, chemical modification and mutagenesis studies have identified Glu-98 as the active site nucleophile of nucleoside 2-deoxyribosyltransferase.
authors.affiliation: Division of Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Porter
authors.forename: B M
authors.initials: BM
authors.lastname: Merrill
authors.forename: S A
authors.initials: SA
authors.lastname: Short
mesh_terms: D000595:Amino Acid Sequence; D001087:Arabinonucleosides / Q000494:pharmacology; D001665:Binding Sites; D018698:Glutamic Acid; D007700:Kinetics; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry*
chemical_list: D001087:Arabinonucleosides; D018698:Glutamic Acid; D010430:Pentosyltransferases; C021888:nucleoside deoxyribosyltransferase