Relationship between the catalytic sites of human bifunctional IMP synthase.
OBJECTIVE
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]AICAR formed from [3H]AICAR indicating that the channelling of FAICAR was not absolute.


CONCLUSIONS
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 if dimeric, bifunctional IMP synthase.
Szabados
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E
Department of Biochemistry, University of Sydney, NSW, Australia.
Christopherson
R I
RI
D001665:Binding Sites; D002384:Catalysis; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases / Q000378:metabolism*; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D009709:Nucleotide Deaminases / Q000378:metabolism*; D011687:Purines; D012265:Ribonucleotides / Q000378:metabolism
D009097:Multienzyme Complexes; D011687:Purines; D012265:Ribonucleotides; C113120:inosine monophosphate synthase; C062030:5-formamidoimidazole-4-carboxamide ribotide; D019877:Hydroxymethyl and Formyl Transferases; C021561:methionyl-tRNA formyltransferase; D009709:Nucleotide Deaminases; C021436:IMP cyclohydrolase