Boyd Jeffrey M, Ensign Scott A
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, USA.
Biochemistry. 2005 Jun 14;44(23):8543-53. doi: 10.1021/bi050393k.
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 on CO(2). In the present study alpha-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, Mg(2+), and a monovalent cation (K(+), Rb(+), NH(4)(+)), and produced mixtures of isotopomers, ranging from singly exchanged H(1)D(5)- 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 stoichiometric 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 K(m) 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 (H(2)O vs D(2)O) 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 CO(2).
丙酮羧化酶催化丙酮羧化生成乙酰乙酸，同时伴随ATP水解生成AMP和两个无机磷酸。丙酮羧化酶的生化、分子和遗传特性表明它代表了一类全新的羧化酶。作为催化的第一步，从一个固有碱性（pK(a)=20）的甲基上夺取一个α-质子，以生成用于进攻CO(2)的必要碳负离子。在本研究中，通过使用气相色谱/质谱法跟踪D(6)-丙酮与水之间的质子-氘核交换，对丙酮的α-质子夺取进行了研究。丙酮羧化酶催化的质子-氘核交换依赖于ATP、Mg(2+)和一价阳离子（K(+)、Rb(+)、NH(4)(+)）的存在，并产生了同位素异构体的混合物，范围从单交换的H(1)D(5)-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)。同位素交换的时间进程表明，每次丙酮结合事件都会发生多次交换事件，并且水解的ATP分子与形成的新丙酮同位素异构体总和之间存在1:1的化学计量关系。在同位素交换过程中，形成的主要ATP水解产物是ADP而不是AMP。K(+)对H(+)(-)D(+)交换和ATP水解的刺激遵循饱和动力学，两种活性的表观K(m)值分别为13.6和14.2 mM。H(+)交换到D(6)-丙酮中的速率大于D(+)交换到H(6)-丙酮中的速率。对于丙酮羧化存在可观察到的溶剂（H(2)O与D(2)O）同位素效应（1.7），但没有可辨别的底物（H(6)-与D(6)-丙酮）同位素效应。有人提出，从丙酮中夺取α-质子与ATP的γ-磷酸基团转移到羰基氧上同时发生，生成磷酸烯醇丙酮作为进攻CO(2)的活化亲核试剂。
