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.