Catalytic mechanism of the cyclohydrolase activity of human aminoimidazole carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase.
The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is responsible for catalysis of the last two steps in the de novo purine pathway. Using recently determined crystal structures of ATIC as a guide, four candidate residues, Lys66, Tyr104, Asp125, and Lys137, were identified for site-directed mutagenesis to study the cyclohydrolase activity of this bifunctional enzyme. Steady-state kinetic experiments on these mutants have shown that none of these residues are absolutely required for catalytic activity; however, they strongly influence the efficiency of the reaction. Since the FAICAR binding site is made up mostly of backbone interactions with highly conserved residues, we postulate that these conserved interactions orient FAICAR in the active site to favor the intramolecular ring closure reaction and that this reaction may be catalyzed by an orbital steering mechanism. Furthermore, it was shown that Lys137 is responsible for the increase in cyclohydrolase activity for dimeric ATIC, which was reported previously by our laboratory. From the experiments presented here, a catalytic mechanism for the cyclohydrolase activity is postulated.
Vergis
James M
JM
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Beardsley
G Peter
GP
D000409:Alanine / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D001717:Birds; D002384:Catalysis; D041961:Deuterium Exchange Measurement; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009709:Nucleotide Deaminases / Q000737:chemistry* / Q000235:genetics; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D014443:Tyrosine / Q000235:genetics
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D001224:Aspartic Acid; D014443:Tyrosine; D001120:Arginine; D019877:Hydroxymethyl and Formyl Transferases; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D009709:Nucleotide Deaminases; C021436:IMP cyclohydrolase; D008239:Lysine; D000409:Alanine