Laseke Amanda J, Lohman Jeremy R, St Maurice Martin
Department of Biological Sciences, Marquette University, Milwaukee, WI, 53201-1881, USA.
Department of Biochemistry and Molecular Biology, Michigan State University, 603 Wilson Road, East Lansing, MI, 48824, USA.
Arch Biochem Biophys. 2025 Feb;764:110280. doi: 10.1016/j.abb.2024.110280. Epub 2024 Dec 24.
Pyruvate carboxylase (PC) catalyzes the carboxylation of pyruvate to oxaloacetate which serves as an important anaplerotic reaction to replenish citric acid cycle intermediates. In most organisms, the PC-catalyzed reaction is allosterically activated by acetyl-coenzyme A. It has previously been reported that vertebrate PC can catalyze the hydrolysis of acetyl-CoA, offering a potential means for the enzyme to attenuate its allosteric activation. However, in the years since this initial report, there has been no further investigation of this phenomenon. The allosteric binding site for acetyl-CoA is now well characterized, enabling more detailed studies on acetyl-CoA hydrolysis at the allosteric site. Here, we confirm that slow acetyl-CoA hydrolysis is catalyzed by a bacterial PC from Staphylococcus aureus, indicating that this phenomenon is a broad feature of PC enzymes spanning the domains of life. Surprisingly, the enzyme can hydrolyze acetyl-CoA even when the binding site for the acetyl moiety is eliminated through truncation of the biotin carboxylase domain. This suggests that an alternative site for acetyl-CoA binding and hydrolysis may be present in the carboxyltransferase domain of S. aureus PC. We conclude that PC has evolved to minimize the rate of acetyl-CoA hydrolysis at the allosteric site and update the description of PC-catalyzed acetyl-CoA hydrolysis to suggest that this reaction is unlikely to play a significant physiological, metabolic or catalytic role.
丙酮酸羧化酶（PC）催化丙酮酸羧化生成草酰乙酸，这是一种重要的回补反应，用于补充柠檬酸循环中间体。在大多数生物体中，PC催化的反应受到乙酰辅酶A的变构激活。此前有报道称，脊椎动物的PC可以催化乙酰辅酶A的水解，这为该酶减弱其变构激活提供了一种潜在方式。然而，自最初报道以来的这些年里，尚未对这一现象进行进一步研究。现在乙酰辅酶A的变构结合位点已得到充分表征，这使得对变构位点处乙酰辅酶A水解的更详细研究成为可能。在这里，我们证实来自金黄色葡萄球菌的一种细菌PC能催化缓慢的乙酰辅酶A水解，这表明这种现象是跨越生命域的PC酶的一个广泛特征。令人惊讶的是，即使通过截短生物素羧化酶结构域消除了乙酰部分的结合位点，该酶仍能水解乙酰辅酶A。这表明在金黄色葡萄球菌PC的羧基转移酶结构域中可能存在乙酰辅酶A结合和水解的替代位点。我们得出结论，PC已经进化以最小化变构位点处乙酰辅酶A的水解速率，并更新了对PC催化的乙酰辅酶A水解的描述，表明该反应不太可能发挥显著的生理、代谢或催化作用。
