Thakkar J K, Janero D R, Sharif H M, Hreniuk D, Yarwood C
Research Department, Ciba Pharmaceuticals, Summit, NJ 07901.
Biochem J. 1994 Jun 1;300 ( Pt 2)(Pt 2):359-63. doi: 10.1042/bj3000359.
Adenylate deaminase (EC 3.5.4.6) may help to regulate the adenine nucleotide catabolism characteristic of such disease states as myocardial ischaemia. We report analysis of the molecular, kinetic and allosteric properties of rabbit heart adenylate deaminase when extracted and purified under phosphate-free conditions (i.e., with Hepes/KOH). The enzyme's subunit molecular mass (approximately 81 kDa), pI (6.5), substrate specificity for 5'-AMP, and activation by K+ were identical in the absence or presence of phosphate. At each chromatographic step during isolation without phosphate, cardiac adenylate deaminase showed a lower apparent activity as compared with the enzyme prepared with phosphate present. Kinetic constants for the phosphate-free rabbit heart adenylate deaminase preparation (Km 0.54 mM AMP; Vmax. 1.4 mumol/min per mg of protein) were approximately 10-fold lower than those of the enzyme isolated with phosphate. The same irreversible decrease in kinetic constants could be achieved by dialysing phosphate from the phosphate-containing enzyme preparation. The relationship between enzyme activity and substrate concentration was sigmoidal in the presence of phosphate, but hyperbolic in its absence. Cardiac adenylate deaminase under phosphate-free conditions was no longer allosterically activated by ATP and ADP, yet remained inhibitable by GTP. Enzyme inhibition by the transition-state mimic coformycin was not influenced by phosphate status. The phosphate-free preparation of rabbit heart adenylate deaminase was markedly labile and extremely susceptible to proteolysis by trypsin or chymotrypsin. The inactivation kinetics and fragmentation pattern in response to controlled proteolysis depended on whether the enzyme had been isolated with or without phosphate present, suggesting a conformational difference between the two enzyme preparations. These data constitute direct evidence that the absence of phosphate irreversibly converts cardiac adenylate deaminase into a pseudo-isoenzyme with distinct kinetic, regulatory and stability properties.
腺苷酸脱氨酶（EC 3.5.4.6）可能有助于调节诸如心肌缺血等疾病状态下特有的腺嘌呤核苷酸分解代谢。我们报告了在无磷酸盐条件下（即使用Hepes/KOH）提取和纯化兔心脏腺苷酸脱氨酶时，对其分子、动力学和别构性质的分析。在有无磷酸盐的情况下，该酶的亚基分子量（约81 kDa）、pI（6.5）、对5'-AMP的底物特异性以及K+的激活作用均相同。在无磷酸盐的分离过程中的每个色谱步骤中，与存在磷酸盐时制备的酶相比，心脏腺苷酸脱氨酶的表观活性较低。无磷酸盐的兔心脏腺苷酸脱氨酶制剂的动力学常数（Km 0.54 mM AMP；Vmax. 1.4 μmol/分钟每毫克蛋白质）比用磷酸盐分离的酶低约10倍。通过从含磷酸盐的酶制剂中透析去除磷酸盐，也可实现动力学常数同样的不可逆降低。在有磷酸盐存在时，酶活性与底物浓度之间的关系呈S形，但在无磷酸盐时呈双曲线形。无磷酸盐条件下的心脏腺苷酸脱氨酶不再被ATP和ADP别构激活，但仍可被GTP抑制。过渡态类似物助间型霉素对酶的抑制作用不受磷酸盐状态的影响。无磷酸盐的兔心脏腺苷酸脱氨酶制剂明显不稳定，极易被胰蛋白酶或糜蛋白酶水解。响应可控蛋白水解的失活动力学和片段化模式取决于酶是在有无磷酸盐的情况下分离得到的，这表明两种酶制剂之间存在构象差异。这些数据构成了直接证据，即无磷酸盐会不可逆地将心脏腺苷酸脱氨酶转化为具有独特动力学、调节和稳定性特性的假同工酶。
