Merkler D J, Wali A S, Taylor J, Schramm V L
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
J Biol Chem. 1989 Dec 15;264(35):21422-30.
Eukaryotes have been proposed to depend on AMP deaminase as a primary step in the regulation of intracellular adenine nucleotide pools. This report describes 1) the role of AMP deaminase in adenylate metabolism in yeast cell extracts, 2) a method for large scale purification of the enzyme, 3) the kinetic properties of native and proteolyzed enzymes, 4) the kinetic reaction mechanism, and 5) regulatory interactions with ATP, GTP, MgATP, ADP, and PO4. Allosteric regulation of yeast AMP deaminase is of physiological significance, since expression of the gene is constitutive (Meyer, S. L., Kvalnes-Krick, K. L., and Schramm, V. L. (1989) Biochemistry 28, 8734-8743). The metabolism of ATP in cell-free extracts of yeast demonstrates that AMP deaminase is the sole pathway of AMP catabolism in these extracts. Purification of the enzyme from bakers' yeast yields a proteolytically cleaved enzyme, Mr 86,000, which is missing 192 amino acids from the N-terminal region. Extracts of Escherichia coli containing a plasmid with the gene for yeast AMP deaminase contained only the unproteolyzed enzyme, Mr 100,000. The unproteolyzed enzyme is highly unstable during purification. Substrate saturation plots for proteolyzed AMP deaminase are sigmoidal. In the presence of ATP, the allosteric activator, the enzyme exhibits normal saturation kinetics. ATP activates the proteolyzed AMP deaminase by increasing the affinity for AMP from 1.3 to 0.2 mM without affecting VM. Activation by ATP is more efficient than MgATP, with half-maximum activation constants of 6 and 80 microM, respectively. The kinetic properties of the proteolyzed and unproteolyzed AMP deaminase are similar. Thus, the N-terminal region is not required for catalysis or allosteric activation. AMP deaminase is competitively inhibited by GTP and PO4 with respect to AMP. The inhibition constants for these inhibitors decrease in the presence of ATP. ATP, therefore, tightens the binding of GTP, PO4, and AMP. The products of the reaction, NH3 and IMP, are competitive inhibitors against substrate, consistent with a rapid equilibrium random kinetic mechanism. Kinetic dissociation constants are reported for the binary and ternary substrate and product complexes and the allosteric modulators.
真核生物被认为依赖于AMP脱氨酶作为调节细胞内腺嘌呤核苷酸池的第一步。本报告描述了：1）AMP脱氨酶在酵母细胞提取物中腺苷酸代谢中的作用；2）一种大规模纯化该酶的方法；3）天然酶和经蛋白酶解的酶的动力学性质；4）动力学反应机制；5）与ATP、GTP、MgATP、ADP和PO4的调节相互作用。酵母AMP脱氨酶的变构调节具有生理意义，因为该基因的表达是组成型的（Meyer, S. L., Kvalnes-Krick, K. L., and Schramm, V. L. (1989) Biochemistry 28, 8734 - 8743）。酵母无细胞提取物中ATP的代谢表明，AMP脱氨酶是这些提取物中AMP分解代谢的唯一途径。从面包酵母中纯化该酶得到一种经蛋白酶解的酶，分子量为86,000，其N端区域缺失192个氨基酸。含有酵母AMP脱氨酶基因质粒的大肠杆菌提取物中仅含有未被蛋白酶解的酶，分子量为100,000。未被蛋白酶解的酶在纯化过程中高度不稳定。经蛋白酶解的AMP脱氨酶的底物饱和曲线呈S形。在变构激活剂ATP存在下，该酶表现出正常的饱和动力学。ATP通过将对AMP的亲和力从1.3 mM提高到0.2 mM来激活经蛋白酶解的AMP脱氨酶，而不影响Vmax。ATP的激活比MgATP更有效，半最大激活常数分别为6和80 μM。经蛋白酶解的和未被蛋白酶解的AMP脱氨酶的动力学性质相似。因此，N端区域对于催化或变构激活不是必需的。AMP脱氨酶相对于AMP受到GTP和PO4的竞争性抑制。在ATP存在下，这些抑制剂的抑制常数降低。因此，ATP增强了GTP、PO4和AMP的结合。反应产物NH3和IMP是底物的竞争性抑制剂，这与快速平衡随机动力学机制一致。报告了二元和三元底物及产物复合物以及变构调节剂的动力学解离常数。
