Evidence Supporting Substrate Channeling between Domains of Human PAICS: A Time-Course Analysis of 13C-Bicarbonate Incorporation.

Abstract

Human phosphoribosylaminoimidazole carboxylase phosphoribosylaminoimdiazole succinocarboxamide synthetase (PAICS) is a dual activity enzyme catalyzing two consecutive reactions in de novo purine nucleotide synthesis. Crystallographic structures of recombinant human PAICS suggested the channeling of 4-carboxy-5-aminoimidazole-1-ribose-5'-phosphate (CAIR) between two active sites of PAICS, while a prior work of an avian PAICS suggested otherwise. Here, we present time-course mass spectrometric data supporting the channeling of CAIR between domains of recombinant human PAICS. Time-course mass spectral analysis showed that CAIR added to the bulk solution (CAIRbulk) is decarboxylated and re-carboxylated before the accumulation of succinyl-5-aminoimidazole-4-carboxamide-1-ribose-5'-phosphate (SAICAR). An experiment with 13C-bicarbonate showed that SAICAR production was proportional to re-carboxylated CAIR instead of total CAIR or CAIRbulk. This result indicates that the SAICAR synthase domain selectively uses enzyme-made CAIR over CAIRbulk, which is consistent with the channeling model. This channeling between PAICS domains may be a part of a larger channeling process in de novo purine nucleotide synthesis.