Porter D J, Spector T
Experimental Therapy Division, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1993 Feb 5;268(4):2480-5.
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.
腺苷脱氨酶（ADA）在腺苷及四种腺苷类似物的脱氨过程中，其蛋白质荧光受到干扰。通过与酶中间体形成相关的荧光变化，监测了周转过程中达到稳态的动力学。这些动力学数据和稳态动力学数据根据以下动力学方案进行分析。[公式：见正文]稳态周转数被指定为k2，腺苷的k2为244 s-1，6-甲基氨基-2-氨基嘌呤阿拉伯糖苷（aMDAP）的k2为1.1×10-3 s-1。结合速率常数k1和解离速率常数k-1的值根据达到稳态的动力学计算得出。k1从腺苷的31×106 M-1 s-1变化到N-6-甲基腺嘌呤阿拉伯糖苷的2.8×106 M-1 s-1。k-1从腺苷的500 s-1变化到aMDAP的31 s-1。通过用过量的赤藓红-9-(2-羟基-3-壬基)腺嘌呤作为aMDAP对ADA进行光谱荧光监测，证实了后者的值（22 s-1）。ADA解离。决定催化作用的k2与k-1的比值从腺苷的0.49降至aMDAP的3.5×10-5。根据k1、k-1和k2计算出的Km值与从稳态动力学数据确定的值相似。酶结合的aMDAP的光谱类似于质子化的aMDAP。
