N-1-(5 '-phosphoribosyl)adenosine-5 '-monophosphate cyclohydrolase: Purification and characterization of a unique metalloenzyme

N-1-(5'-Phosphoribosyl)adenosine-5'-monophosphate cyclohydrolase (HisI, PR- AMP cyclohydrolase) is a central enzyme in histidine biosynthesis catalyzin g the hydrolysis of the N1-C6 bond of the purine substrate, a reaction uniq ue to this pathway. A source of the recombinant monofunctional Methanococcu s vannielii PR-AMP cyclohydrolase has been developed, and the first charact erization of a purified form of the enzyme is reported. The enzyme has a na tive molecular weight of 31 200 as determined by analytical ultracentrifuga tion that agrees with the molecular mass determined by gel filtration (34 k Da) 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 con firms their origin as PR-AMP cyclohydrolase. A highly conserved region of t he amino acid sequence is implicated in the self-cleavage events of the pro tein 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 dialy sis with 1,10-phenanthroline (K-d less than or equal to 10(-9) M). Removal of the Zn2+ correlates with the loss of enzyme activity. The enzyme is reve rsibly inhibited by inclusion of EDTA in the assay mixture, demonstrating t hat free Mg2+ (K-s = 4.9 +/- 0.7 mu M) is required for catalytic activity. Further evidence for a low-affinity binding site is indicated by the inhibi tory effects of exogenous Zn2+ on enzyme activity. The pH dependence of the PR-AMP cyclohydrolase activity shows a single titration event in the k(cat )/K-m profile with a pK(a) of 7.3 that is consistent with the functional ro le of a metal site in catalysis. These data are discussed in the context of the mechanism of other nucleotide hydrolases.