Nucleoside 2-deoxyribosyltransferase. Pre-steady-state kinetic analysis of native enzyme and mutant enzyme with an alanyl residue replacing Glu-98.
Nucleoside 2-deoxyribosyltransferase catalyzes cleavage of a 2'-deoxyribosylnucleoside (A) to a nucleobase (P) with deoxyribosylation of the enzyme. Substrates quenched the intrinsic fluorescence of native enzyme (E) and a catalytically inactive mutant enzyme (E98A enzyme). The time courses of these reactions were analyzed in terms of the following scheme where EX is the 2-deoxyribosyl ester of Glu-98. [formula: see text] The initial complexes between E and dAdo, dGuo, dIno, and dCyd or those between EX and the corresponding nucleobases were formed in a rapid equilibrium step. Native enzyme and E98A enzyme bound 2'-deoxyribosylnucleosides with similar affinities (k-1/k1). From a comparison of the time-dependent fluorescence changes associated with the reaction of native enzyme or E98A enzyme with these substrate, the kinetic step for 2-deoxyribosylation of Glu-98 was identified (k2 and k-2). dThd and dUrd quenched the fluorescence of native enzyme in a biphasic process. The late phase of this reaction was associated with 2-deoxyribosylation of Glu-98. The pre-steady-state kinetic constants calculated from fluorescence quenching data for dAdo and Cyt were consistent with the experimental values for the steady-state kinetic coefficients and the equilibrium constant of the reaction.
Porter
D J
DJ
Division of Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709, USA.
Short
S A
SA
D000225:Adenine / Q000378:metabolism; D000409:Alanine; D003839:Deoxyadenosines / Q000378:metabolism; D003857:Deoxyuridine / Q000378:metabolism; D005453:Fluorescence; D018698:Glutamic Acid; D007700:Kinetics; D009154:Mutation; D010430:Pentosyltransferases / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013936:Thymidine / Q000378:metabolism
D003839:Deoxyadenosines; D018698:Glutamic Acid; D010430:Pentosyltransferases; C021888:nucleoside deoxyribosyltransferase; D000225:Adenine; D000409:Alanine; C058118:2'-deoxyadenosine; D013936:Thymidine; D003857:Deoxyuridine