ATP-dependent enolization of acetone by acetone carboxylase from Rhodobacter capsulatus
ATP-dependent enolization of acetone by acetone carboxylase from Rhodobacter capsulatus
复制标题
DOI：
10.1021/bi050393k
复制
发表时间：
2005-06-14
期刊：
影响因子：
2.9
通讯作者：
Ensign, SA
中科院分区：
文献类型：
--
作者：
Boyd, JM;Ensign, SA
丙酮羧化酶催化丙酮羧化为乙酰乙酸，同时ATP水解为AMP和两种无机磷酸盐。丙酮羧化酶的生物化学、分子和遗传特性表明它代表了一类全新的羧化酶。作为催化的初始步骤，从固有碱性（pK（a）= 20）甲基中提取α-质子以产生用于攻击油CO2的必需碳负离子。在本研究中，α-质子提取丙酮已被调查，通过使用气相色谱/质谱遵循质子-氘交换D-6-丙酮和水之间。丙酮羧化酶催化的质子-氘交换依赖于ATP、Mg 2+和一价阳离子（K+、Rb+、NH 4+）的存在，并产生同位素异构体的混合物，范围从单交换的H1 D5-到完全交换的H-6-丙酮。同位素交换的初始速率高于丙酮羧化的k（cat）。同位素交换的时间过程表明，多个交换事件发生的每个丙酮结合事件，并有1：1的化学计量关系之间的ATP水解的分子和新的丙酮同位素形成的总和。ADP而不是AMP是同位素交换过程中ATP水解的主要产物。H+-D+交换和ATP水解的刺激K+遵循饱和动力学，这两种活动的表观Km值分别为13.6和14.2 mM。H+交换成D-6-丙酮的速率大于D+交换成H-6-丙酮的速率。丙酮羧化反应存在可观察到的溶剂（H2O vs D2 O）同位素效应（1.7），但没有明显的底物（H-6- vs D-6-丙酮）同位素效应。有人提出，从丙酮中的α-质子提取发生在音乐会与ATP的γ-磷酰基转移到羰基氧，生成磷酸烯醇丙酮作为攻击CO2的活化亲核试剂。
Acetone carboxylase catalyzes the carboxylation of acetone to acetoacetate with concomitant hydrolysis of ATP to AMP and two inorganic phosphates. The biochemical, molecular, and genetic properties of acetone carboxylase suggest it represents a fundamentally new class of carboxylase. As the initial step in catalysis, an alpha-proton from an inherently basic (pK(a) = 20) methyl group is abstracted to generate the requisite carbanion for attack oil CO2. In the present study cc-proton abstraction from acetone has been investigated by using gas chromatography/mass spectrometry to follow proton-deuteron exchange between D-6-acetone and water. Acetone carboxylase-catalyzed proton-deuteron exchange was dependent upon the presence of ATP, Mg2+, and a monovalent cation (K+, Rb+, NH4+), and produced mixtures of isotopomers, ranging from singly exchanged H1D5- to fully exchanged H-6-acetone. The initial rate of isotopic exchange was higher than k(cat) for acetone carboxylation. The time course of isotopic exchange showed that multiple exchange events occur for each acetone-binding event, and there was a 1:1 stoichiomettic relationship between molecules of ATP hydrolyzed and the sum of new acetone isotopomers formed. ADP rather than AMP was formed as the predominant product of ATP hydrolysis during isotopic exchange. The stimulation of H+-D+ exchange and ATP hydrolysis by K+ followed saturation kinetics, with apparent Km values of 13.6 and 14.2 mM for the two activities, respectively. The rate of H+ exchange into D-6-acetone was greater than the rate of D+ exchange into H-6-acetone. There was an observable solvent (H2O vs D2O) isotope effect (1.7) for acetone carboxylation but no discernible substrate (H-6- vs D-6-acetone) isotope effect. It is proposed that alpha-proton abstraction from acetone occurs in concert with transfer of the gamma-phosphoryl group of ATP to the carbonyl oxygen, generating phosphoenol acetone as the activated nucleophile for attack on CO2.