The Molecular Environment Modulates CO2 Liberation from Carboxy-Biotin

Carboxy-biotin serves as a coenzyme in certain carboxylases, exhibiting the remark- able capability to transfer a carboxy group to specific substrates. This process is made possible by the presence of biotin, a unique molecule that consists of a sulfur-containing tetrahydrothiophene ring fused to a ureido group. It is covalently attached to the en- zyme via a flexible linker allowing for its functionality. Biotin-dependent carboxylases consist of two distinct domains. The first domain facilitates biotin carboxylation by utilizing ATP, while the second domain transfers CO2 to the substrate. The process of ATP-dependent carboxylation using bicarbonate in the biotin carboxylase domain is well known. However, the precise mechanism by which CO2 is released in the carboxy- transferase domain is still not fully understood. We employed advanced computational chemistry methods to investigate the decarboxylation process of carboxy-biotin in vari- ous molecular environments and different protonation states. Regardless of the polarity of the molecular surroundings, decarboxylation only occurs spontaneously in the pro- tonated form. To determine the protonation state of biotin in different environments, we established an accurate computational chemistry method for calculating the pKa value of carboxy-biotin reaching sub-kcal mol−1 accuracy. Based on our findings, non- polar environments, such as the active site of the carboxy-transferase domain, have the ability to cause the spontaneous release of CO2 from carboxy-biotin. The CO2 release takes place spontaneously from protonated carboxy-biotin promoting the carboxylation of substrates.