Alternative substrates for calf intestinal adenosine deaminase. A pre-steady-state kinetic analysis.

The protein fluorescence of adenosine deaminase (ADA) was perturbed during the deamination of adenosine and four analogues of adenosine. The kinetics for the approach to the steady-state during turnover were monitored by fluorescence changes associated with formation of enzymatic intermediates. These kinetic data and the steady-state kinetic data were analyzed in terms of the kinetic scheme as follows. [formula: see text] The steady-state turnover number was assigned to k2, which was 244 s-1 for adenosine and 1.1 x 10(-3) s-1 for 6-methylamino-2-aminopurine arabinoside (aMDAP). Values for the association rate constants, k1, and the dissociation rate constants, k-1, were calculated from the kinetics for the approach to the steady state. k1 varied from 31 x 10(6) M-1 s-1 for adenosine to 2.8 x 10(6) M-1 s-1 for N-6-methyladenine arabinoside. k-1 varied from 500 s-1 for adenosine to 31 s-1 for aMDAP. The latter value was confirmed (22 s-1) by spectrofluorometrically monitoring the trapping of ADA by excess erythro-9-(2-hydroxy-3-nonyl) adenine as aMDAP.ADA dissociated. The ratio of k2 to k-1, which determines the commitment to catalysis, decreased from 0.49 for adenosine to 3.5 x 10(-5) for aMDAP. The Km values calculated from k1, k-1, and k2 were similar to the values determined from steady-state kinetic data. The spectrum of enzyme-bound aMDAP resembled protonated aMDAP.