Relationship between the catalytic sites of human bifunctional IMP synthase

Background and Aims: The bifunctional enzyme, IMP synthase, contains 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase and IMP cyclohydrolase activities and catalyses the ninth and tenth reactions of the pathway for de novo biosynthesis of purine nucleotides (AICAR→FAICAR→IMP). The spatial relationship between the two active sites on IMP synthase has been investigated along with the possibility that the intermediate, FAICAR, may be channelled between the two sites. Methods: The two catalytic activities and the overall reaction (AICAR→FAICAR→IMP) were assayed using 3H-labelled AICAR or FAICAR with isolation of the reaction products by thin-layer chromatography. Results: Inhibition constants for the interactions of six purine nucleoside 5′-monophosphate derivatives with AICAR transformylase and IMP cyclohydrolase were 24- to 820-fold higher for the transformylase. N-ethylmaleimide inactivated IMP cyclohydrolase but not AICAR transformylase. The rate of IMP synthesis from AICAR was consistent with a high local concentration of FAICAR at the cyclohydrolase site but addition of exogenous unlabelled FAICAR reduced the amount of [ 3H]IMP formed from [ 3H]AICAR indicating that the channelling of FAICAR was not absolute. Conclusion: The AICAR transformylase and IMP cyclohydrolase active sites of IMP synthase are distinct but sufficiently close for the FAICAR produced by a transformylase site to be preferentially utilized as a substrate by a cyclohydrolase site on the same molecule of dimeric, bifunctional IMP synthase.