Catalytic zinc site and mechanism of the metalloenzyme PR-AMP cyclohydrolase

Abstract

The enzyme N(1)-(5'-phosphoribosyl) adenosine-5'-monophosphate cyclohydrolase (PR-AMP cyclohydrolase) is a Zn(2+) metalloprotein encoded by the hisI gene. It catalyzes the third step of histidine biosynthesis, an uncommon ring-opening of a purine heterocycle for use in primary metabolism. A three-dimensional structure of the enzyme from Methanobacterium thermoautotrophicum has revealed that three conserved cysteine residues occur at the dimer interface and likely form the catalytic site. To investigate the functions of these cysteines in the enzyme from Methanococcus vannielii, a series of biochemical studies were pursued to test the basic hypothesis regarding their roles in catalysis. Inactivation of the enzyme activity by methyl methane thiosulfonate (MMTS) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) also compromised the Zn(2+) binding properties of the protein inducing loss of up to 90% of the metal. Overall reaction stoichiometry and the potassium cyanide (KCN) induced cleavage of the protein suggested that all three cysteines were modified in the process. The enzyme was protected from DTNB-induced inactivation by inclusion of the substrate N(1)-(5'-phosphoribosyl)adenosine 5'-monophosphate; (PR-AMP), while Mg(2+), a metal required for catalytic activity, enhanced the rate of inactivation. Site-directed mutations of the conserved C93, C109, C116 and the double mutant C109/C116 were prepared and analyzed for catalytic activity, Zn(2+) content, and reactivity with DTNB. Substitution of alanine for each of the conserved cysteines showed no measurable catalytic activity, and only the C116A was still capable of binding Zn(2+). Reactions of DTNB with the C109A/C116A double mutant showed that C93 is completely modified within 0.5 s. A model consistent with these data involves a DTNB-induced mixed disulfide linkage between C93 and C109 or C116, followed by ejection of the active site Zn(2+) and provides further evidence that the Zn(2+) coordination site involves the three conserved cysteine residues. The C93 reactivity is modulated by the presence of the Zn(2+) and Mg(2+) and substantiates the role of this residue as a metal ligand. In addition, Mg(2+) ligand binding site(s) indicated by the structural analysis were probed by site-directed mutagenesis of three key aspartate residues flanking the conserved C93 which were shown to have a functional impact on catalysis, cysteine activation, and metal (zinc) binding capacity. The unique amino acid sequence, the dynamic properties of the cysteine ligands involved in Zn(2+) coordination, and the requirement for a second metal (Mg(2+)) are discussed in the context of their roles in catalysis. The results are consistent with a Zn(2+)-mediated activation of H(2)O mechanism involving histidine as a general base that has features similar to but distinct from those of previously characterized purine and pyrimidine deaminases.

Figure 1

Highly Conserved Regions of PR-AMP Cyclohydrolase. A.) Alignment highlights twelve of the most highly conserved residues in the cyclohydrolase enzymes. B.) Pairwise alignment of M. thermoautotrophicum and M. vannielii.

Figure 1

Highly Conserved Regions of PR-AMP Cyclohydrolase. A.) Alignment highlights twelve of the most highly conserved residues in the cyclohydrolase enzymes. B.) Pairwise alignment of M. thermoautotrophicum and M. vannielii.

Figure 2

MMTS Inactivation of PR-AMP Cyclohydrolase and Effect of Substrate on Reactivity of Cysteines. Data are as follows (△) not determined, control no MMTS; (●) 1.7×10⁻² min⁻¹, 78 μM MMTS, 1.1 mM PR-AMP; (□) 4.8 x10⁻² min⁻¹, 39 μM MMTS; (■) 1.5 X10⁻¹ min⁻¹, 59 μM MMTS; (○) 2.6×10⁻¹ min⁻¹, 78 μM MMTS.

Figure 3

DTNB Inactivation Rates of PR-AMP Cyclohydrolase and the Effect of PR-AMP and Mg². Data are as follows A. (△) 2.7×10⁻³ min⁻¹, control no DTNB, (○) 8.5×10⁻² min^−1, 212 μM DTNB, (▲)1.82 x10⁻² min⁻¹, 212 μM DTNB with 1.0 mM PR-AMP; B. (◆) 2.7×10⁻³ min⁻¹, control no DTNB, (○) 1.6×10⁻² min⁻¹, in the absence of Mg²⁺, 424 μM DTNB, (■) 2.5×10⁻¹ min⁻¹, 424 μM DTNB.

Figure 4

Stoichiometry and Kinetics of Cyclohydrolase Reaction with DTNB. A.) PR-AMP cyclohydrolase complete reaction with DTNB. Conditions 50 mM Tris-HCl pH 8.5, 1mM EDTA, 5 mM MgCl₂ 30°C 625 μM DTNB, and 12.5 μM PR-AMP cyclohydrolase. B.) Stopped-flow results for the reaction of DTNB with native PR-AMP cyclohydrolase (solid line), cysteine (dots), and C109/116A (dashes). Conditions 50 mM KH₂PO₄ pH 7.3, 0.5 mM EDTA 1mM MgCl₂ 20ºC 440 μM DTNB 8.7 μM PR-AMP cyclohydrolase.

Figure 5

Stopped flow analysis of the PR-AMP Cyclohydrolase enzymatic conversion: Plots (averages of triplicate runs) of the time vs. concentration for substrate, product and potential intermediate from global fit of the data to a two-step model A>B>C. Open circles (○) are substrate(S), closed circles (●) are intermediate (I), and open triangles (△) are product (P) as labeled.

Figure 6

A structural model of the PR-AMP cyclohydrolase active site with substrate docked. (A) The metal Zn²⁺ and Mg²⁺ binding sites and their relationships to the conserved amino acid residues are indicated to assess the functional roles in substrate binding site. The 16 residues labeled in the figure are among the most highly conserved residues described in Figure 1 and reside within 4 Å of the substrate binding site. (B) To better understand the potential positioning of the active site for a zinc-activated water attack on the C6 of the purine ring, a molecule of water was manually positioned into the substrate-docked model. Distances (black dotted lines) are shown in angstroms (Å). Hydrogen bonds and coordinative bonds to metals are rendered as colored spheres.

Figure 6

A structural model of the PR-AMP cyclohydrolase active site with substrate docked. (A) The metal Zn²⁺ and Mg²⁺ binding sites and their relationships to the conserved amino acid residues are indicated to assess the functional roles in substrate binding site. The 16 residues labeled in the figure are among the most highly conserved residues described in Figure 1 and reside within 4 Å of the substrate binding site. (B) To better understand the potential positioning of the active site for a zinc-activated water attack on the C6 of the purine ring, a molecule of water was manually positioned into the substrate-docked model. Distances (black dotted lines) are shown in angstroms (Å). Hydrogen bonds and coordinative bonds to metals are rendered as colored spheres.

Scheme 1

Conversion of PR-AMP to 5’-ProFAR by PR-AMP Cyclohydrolase

Scheme 2

Potential Model for Cysteine Reactivity Observed with Native PR-AMP Cyclohydrolase.

Scheme 3

Proposed catalytic mechanism for PR-AMP Cyclohydrolase