Catalytic Cycle of the Bifunctional Enzyme Phosphoribosyl-ATP Pyrophosphohydrolase/Phosphoribosyl-AMP Cyclohydrolase

Abstract

The bifunctional enzyme phosphoribosyl-ATP pyrophosphohydrolase/phosphoribosyl-AMP cyclohydrolase (HisIE) catalyzes the second and third steps of histidine biosynthesis: pyrophosphohydrolysis of N¹-(5-phospho-β-D-ribosyl)-ATP (PRATP) to N¹-(5-phospho-β-D-ribosyl)-AMP (PRAMP) and pyrophosphate in the C-terminal HisE-like domain, and cyclohydrolysis of PRAMP to N-(5'-phospho-D-ribosylformimino)-5-amino-1-(5″-phospho-D-ribosyl)-4-imidazolecarboxamide (ProFAR) in the N-terminal HisI-like domain. Here we use UV-VIS spectroscopy and LC-MS to show Acinetobacter baumannii putative HisIE produces ProFAR from PRATP. Employing an assay to detect pyrophosphate and another to detect ProFAR, we established the pyrophosphohydrolase reaction rate is higher than the overall reaction rate. We produced a truncated version of the enzyme-containing only the C-terminal (HisE) domain. This truncated HisIE was catalytically active, which allowed the synthesis of PRAMP, the substrate for the cyclohydrolysis reaction. PRAMP was kinetically competent for HisIE-catalyzed ProFAR production, demonstrating PRAMP can bind the HisI-like domain from bulk water, and suggesting that the cyclohydrolase reaction is rate-limiting for the overall bifunctional enzyme. The overall k_cat increased with increasing pH, while the solvent deuterium kinetic isotope effect decreased at more basic pH but was still large at pH 7.5. The lack of solvent viscosity effects on k_cat and k_cat/K_M ruled out diffusional steps limiting the rates of substrate binding and product release. Rapid kinetics with excess PRATP demonstrated a lag time followed by a burst in ProFAR formation. These observations are consistent with a rate-limiting unimolecular step involving a proton transfer following adenine ring opening. We synthesized N¹-(5-phospho-β-D-ribosyl)-ADP (PRADP), which could not be processed by HisIE. PRADP inhibited HisIE-catalyzed ProFAR formation from PRATP but not from PRAMP, suggesting that it binds to the phosphohydrolase active site while still permitting unobstructed access of PRAMP to the cyclohydrolase active site. The kinetics data are incompatible with a build-up of PRAMP in bulk solvent, indicating HisIE catalysis involves preferential channeling of PRAMP, albeit not via a protein tunnel.

Scheme 1. HisIE-catalyzed Hydrolysis of PRATP Followed by Ring-Opening Hydrolysis of PRAMP

Figure 1

Substrate saturation curves and associated apparent steady-state kinetic parameters for AbHisIE-catalyzed ProFAR formation and PPi formation. Lines are best fit to eq 1. A Student’s t-test indicated k_cat^ProFAR and k_cat^PPi are statistically different (p < 0.025).

Scheme 2. The Minimum Kinetic Sequence for the AbHisIE-catalyzed Reaction

Figure 2

Kinetic competence of PRAMP. (A) Time courses of ProFAR formation from different PRAMP concentrations. Thick lines are mean traces from two independent measurements; thin black lines are linear regressions of the data. (B) PRAMP saturation curve and associated apparent steady-state kinetic parameters for AbHisIE-catalyzed ProFAR formation. The line is best fit to eq 1.

Figure 3

Solvent viscosity effects on AbHisIE-catalyzed reaction. (A) Substrate saturation curves for AbHisIE-catalyzed ProFAR formation in the presence and absence of glycerol. Lines are best fit to eq 1. (B) Solvent viscosity effects on k_cat^ProFAR/K_M. (C) Solvent viscosity effects on k_cat^ProFAR. Lines are best fit to eq 2.

Figure 4

AbHisIE kinetics in H₂O and D₂O. (A) PRATP saturation curves at pH 7.0, 7.5, and 8.0. Lines are best fit to eq 1. (B) PRATP saturation curves at pD 7.0, 7.5, and 8.0. Lines are best fit to eq 3. (C) Dependence of k_cat^ProFAR/K_M on pL. (D) Dependence of k_cat^ProFAR on pL. (E) PRAMP saturation curves at pL 7. Lines are best fit to eq 1 for data in H₂O and to eq 3 for data in D₂O. L denotes either H or D.

Figure 5

Rapid kinetics of approach to a steady state of AbHisIE-catalyzed ProFAR production from PRATP. The dashed lines are linear regressions of the linear phases.

Figure 6

AbHisIE inhibition by PRADP. (A) Dose-dependence curve of AbHisIE-catalyzed ProFAR formation from PRATP in the presence of PRADP. The line is the best fit to eq 4. (B) Initial rates of AbHisIE-catalyzed ProFAR formation from PRAMP in the presence and absence of PRADP. Thin black lines are linear regressions of the traces which produced initial rates of 0.0565 ± 0.0004 and 0.0534 ± 0.0004 μM s^–1 in the absence and presence of PRADP.

Scheme 3. Expanded Kinetic Sequence for the AbHisIE-Catalyzed Reaction