Analysis of substrate binding in individual active sites of bifunctional human ATIC

Abstract

Aminoimidazolecarboxamide ribonucleotide formyl transferase (AICARFT): Inosine monophosphate cyclohydrolase (IMPCH, collectively called ATIC) is a bifunctional enzyme that catalyses the penultimate and final steps in the purine de novo biosynthesis pathway. The bifunctional protein is dimeric and each monomer contains two different active sites both of which are capable of binding nucleotide substrates, this means to a potential total of four distinct binding events might be observed. Within this work we used a combination of site-directed and truncation mutants of ATIC to independently investigate the binding at these two sites using calorimetry. A single S10W mutation is sufficient to block the IMPCH active site allowing investigation of the effects of mutation on ligand binding in the AICARFT active site. The majority of nucleotide ligands bind selectively at one of the two active sites with the exception of xanthosine monophosphate, XMP, which, in addition to binding in both AICARFT and IMPCH active sites, shows evidence for cooperative binding with communication between symmetrically-related active sites in the two IMPCH domains. The AICARFT site is capable of independently binding both nucleotide and folate substrates with high affinity however no evidence for positive cooperativity in binding could be detected using the model ligands employed in this study.

A surface pocket in the cytoplasmic domain of the herpes simplex virus fusogen gB controls membrane fusion

“…Introducing an amino acid with a bulkier, branched side chain into a pocket on Rabies virus P protein resulted in decreased binding by STAT proteins, suggesting that the mutated pocket constitutes the STAT-binding site [ 56 ]. Furthermore, in enzymes, active sites, which bind and convert substrates, are typically found in surface pockets, and pocket-filling mutations can either block binding of the substrate to the active site [ 57 ] or obstruct its access through a tunnel or channel [ 58 , 59 ].…”

A surface pocket in the cytoplasmic domain of the herpes simplex virus fusogen gB controls membrane fusion

“…Introducing an amino acid with a bulkier, branched side chain into a pocket on Rabies virus P protein resulted in decreased binding by STAT proteins, suggesting that the mutated pocket constitutes the STAT-binding site [52]. Furthermore, in enzymes, active sites, which bind and convert substrates, are typically found in surface pockets, and pocket-filling mutations can either block binding of the substrate to the active site [53] or obstruct its access through a tunnel or channel [54, 55].…”

AICA‐ribosiduria due to ATIC deficiency: Delineation of the phenotype with three novel cases, and long‐term update on the first case

“…Adapted from Reference 13 (EC 2.1.2.3) and IMP cyclohydrolase (EC 3.5.4.10) enzymatic activities, respectively. 7,8 ATIC deficiency results in 5-amino-4-imidazolecarboxamide-ribosiduria (AICA)ribosiduria (OMIM #608688). It is an autosomal recessive disorder described so far in only one patient, presenting with a devastating neurological picture, involving profound intellectual disability, epilepsy, congenital blindness, and dysmorphic features.…”

“…Moreover, PAICS deficiency has been recently reported for the first time in human, in two siblings affected by multiple congenital malformations with fatal neonatal outcome 6 . Finally, bifunctional purine biosynthesis protein PURH (ATIC) is a homodimer which catalyzes the penultimate and final steps of DNPS through its phosphoribosylaminoimidazolecarboxamide formyltransferase (EC 2.1.2.3) and IMP cyclohydrolase (EC 3.5.4.10) enzymatic activities, respectively 7,8 . ATIC deficiency results in 5‐amino‐4‐imidazolecarboxamide‐ribosiduria (AICA)‐ribosiduria (OMIM #608688).…”