title: Structure and functional relationships in human pur H.
abstract: 1. The human pur H (ATIC) gene encoding a bifunctional protein, hPurH, which carries the penultimate and final enzymatic activities of the purine nucleotide synthesis pathway, AICARFT & IMPCH, has been cloned and sequenced. The gene product, hPurH has been overexpressed in E. coli, purified to homogeneity and crystallized. 2. The human pur H gene lies on chromosome 2, between band q34 and q35. There is at least one intron of 278 bp near the 5' end. 3. Truncation mutant studies demonstrate two non-overlapping functional domains in the protein arranged as indicated in Figure 5. The existence of a linker or interaction region between the catalytic domains remains to be established. 4. Cleland-type kinetic inhibition experiments indicate that the AICARFT reaction is of the ordered, sequential type with the reduced folate cofactor binding first. 5. The reaction has a broad pH optimum in the alkaline range, with a maximum at about pH 8.2. 6. Preliminary transient phase kinetic studies show the presence of a "burst" indicating that a late step in the reaction sequence is rate limiting. 7. A PurH crystal structure is that of a dimer, with a putative single binding site for the reduced folate cofactor formed using elements from each of the monomer subunits. Probable binding sites for AICAR and FAICAR can be identified on each monomer. 8. Equilibrium sedimentation studies show hPurH apoprotein to be a monomer:dimer equilibrium mixture with a kD of 0.55 uM. 9. The crystal structure has permitted identification of a number of candidate amino acid residues likely to be involved in catalysis and/or substrate binding. Among these, we have thus far completed studies on two, Lysine 265 and Histidine 266. These appear to be critically involved in the AICARFT reaction, although whether their role(s) are in catalysis or binding remains to be determined.
authors.affiliation: Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520-8064, USA.
authors.forename: G P
authors.initials: GP
authors.lastname: Beardsley
authors.forename: E A
authors.initials: EA
authors.lastname: Rayl
authors.forename: K
authors.initials: K
authors.lastname: Gunn
authors.forename: B A
authors.initials: BA
authors.lastname: Moroson
authors.forename: H
authors.initials: H
authors.lastname: Seow
authors.forename: K S
authors.initials: KS
authors.lastname: Anderson
authors.forename: J
authors.initials: J
authors.lastname: Vergis
authors.forename: K
authors.initials: K
authors.lastname: Fleming
authors.forename: S
authors.initials: S
authors.lastname: Worland
authors.forename: B
authors.initials: B
authors.lastname: Condon
authors.forename: J
authors.initials: J
authors.lastname: Davies
mesh_terms: D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D002874:Chromosome Mapping; D002889:Chromosomes, Human, Pair 2; D003001:Cloning, Molecular; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D009709:Nucleotide Deaminases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D011685:Purine Nucleotides / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012265:Ribonucleotides / Q000378:metabolism
chemical_list: D009097:Multienzyme Complexes; D011685:Purine Nucleotides; D011994:Recombinant Proteins; D012265:Ribonucleotides; C113120:inosine monophosphate synthase; C062030:5-formamidoimidazole-4-carboxamide ribotide; D000620:Aminoimidazole Carboxamide; D019877:Hydroxymethyl and Formyl Transferases; D009709:Nucleotide Deaminases; C031143:AICA ribonucleotide