Active site amino acids that participate in the catalytic mechanism of nucleoside 2'-deoxyribosyltransferase.
The importance of eight nucleoside 2'-deoxyribosyltransferase residues for catalysis was investigated by site-directed mutagenesis. Each residue was selected because of its proximity to nucleophile Glu-98 or on its potential contribution to intrinsic protein fluorescence. Mutation of Asp-72, Asp-92, Tyr-7, Trp-12, and Met-125 resulted in over a 90% activity loss whereas mutation of Tyr-157, Trp-64, and Trp-127 produced less than a 80% activity loss. The magnitude of the perturbation on catalysis by mutation, however, was dependent on donor substrate. The kcat values for dIno hydrolysis by these mutants were greater than 25% of that for native enzyme. Although mutant and native enzymes bound substrate analogues with comparable affinities, Km values for dIno hydrolysis varied over a 1000-fold range. The pH dependence of Glu-98 esterification by dCyd suggested that amino acids with pK values of 4.2 and 7.5 were relevant for catalysis. The intrinsic protein fluorescence was attributed primarily to Trp-127 (approximately 80%). Pre-steady-state kinetic parameters for deoxyribosylation of mutant enzymes by dCyd, dThd, and dAdo were determined by monitoring changes in enzyme fluorescence. Collectively, results from mutagenesis suggest that, depending upon substrate, either Asp-92 or Asp-72 functions as the general acid catalyst, and that this enzyme undergoes a change in conformation upon Glu-98 deoxyribosylation.
Short
S A
SA
Division of Biochemistry, Glaxo-Wellcome, Research Triangle Park, North Carolina 27709, USA.
Armstrong
S R
SR
Ealick
S E
SE
Porter
D J
DJ
D001665:Binding Sites; D002384:Catalysis; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010430:Pentosyltransferases / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D011994:Recombinant Proteins; D010430:Pentosyltransferases; C021888:nucleoside deoxyribosyltransferase