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-β-D-arabinofuranosyl)-9H- purine (dFDAP) inhibited the enzyme by formation of a primary complex (Kd = 140 μM) 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 °C, but regained over 70% of the lost activity by treatment with 600 μM Ade. 6-Amino-9-(2′-deoxy-2′-fluoro-β-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 γ-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.