N1-(5'-phosphoribosyl)adenosine-5'-monophosphate cyclohydrolase: purification and characterization of a unique metalloenzyme.

N1-(5'-Phosphoribosyl)adenosine-5'-monophosphate cyclohydrolase (HisI, PR-AMP cyclohydrolase) is a central enzyme in histidine biosynthesis catalyzing the hydrolysis of the N1-C6 bond of the purine substrate, a reaction unique to this pathway. A source of the recombinant monofunctional Methanococcus vannielii PR-AMP cyclohydrolase has been developed, and the first characterization of a purified form of the enzyme is reported. The enzyme has a native molecular weight of 31 200 as determined by analytical ultracentrifugation that agrees with the molecular mass determined by gel filtration (34 kDa) and a subunit molecular weight of 15 486 based on MALDI-MS. An unusual characteristic of the protein is the complexity observed on SDS-PAGE, and N-terminal amino acid sequence analysis of all the isolated constituents confirms their origin as PR-AMP cyclohydrolase. A highly conserved region of the amino acid sequence is implicated in the self-cleavage events of the protein and provides an explanation for the complexity of this protein. Bound to the enzyme is 1 equiv of Zn2+ that can be removed only by extended dialysis with 1,10-phenanthroline (Kd (1)In general, the qualitative or quantitative analysis of a substance. In MGI, an assay is a type of experiment that is designed to detect the level of gene expression of a particular gene, or to determine the pattern of expression of a gene among different tissue types, anatomical structures, or developmental stages. The assay may detect one or more of the RNA transcripts of a gene or one or more of its protein products. Assay types in MGI include: Immunohistochemistry Northern blot Protein histochemistry RNA In situ hybridization In situ reporter RNAse protection RT-PCR Western blot(2)Analysis to determine the presence, absence, or quantity of one or more components; a test used in this analysis.

" data-original-title="Assay" data-html="true" data-trigger="foucs">assay mixture, demonstrating that free Mg2+ (Ks = 4.9 +/- 0.7 microM) is required for catalytic activity. Further evidence for a low-affinity binding site is indicated by the inhibitory effects of exogenous Zn2+ on enzyme activity. The pH dependence of the PR-AMP cyclohydrolase activity shows a single titration event in the kcat/Km profile with a pKa of 7.3 that is consistent with the functional role of a metal site in catalysis. These data are discussed in the context of the mechanism of other nucleotide hydrolases.

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