The bifunctional
enzyme phosphoribosyl-ATP pyrophosphohydrolase/phosphoribosyl-AMP
cyclohydrolase (HisIE) catalyzes the second and third steps of histidine
biosynthesis: pyrophosphohydrolysis ofN1-(5-phospho-β-D-ribosyl)-ATP (PRATP) toN1-(5-phospho-β-D-ribosyl)-AMP (PRAMP) and pyrophosphate
in the C-terminal HisE-like domain, and cyclohydrolysis of PRAMP toN-(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 showAcinetobacter
baumanniiputative 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 overallkcatincreased 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 onkcatandkcat/KMruled 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 synthesizedN1-(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.