Crystal Structure, Steady-State, and Pre-Steady-State Kinetics of Acinetobacter baumannii ATP Phosphoribosyltransferase

Abstract

The first step of histidine biosynthesis in Acinetobacter baumannii, the condensation of ATP and 5phospho-α-D-ribosyl-1-pyrophosphate to produce N 1 -(5-phosphoβ-D-ribosyl)-ATP (PRATP) and pyrophosphate, is catalyzed by the hetero-octameric enzyme ATP phosphoribosyltransferase, a promising target for antibiotic design. The catalytic subunit, HisG S , is allosterically activated upon binding of the regulatory subunit, HisZ, to form the hetero-octameric holoenzyme (ATPPRT), leading to a large increase in k cat . Here, we present the crystal structure of ATPPRT, along with kinetic investigations of the rate-limiting steps governing catalysis in the nonactivated (HisG S ) and activated (ATPPRT) forms of the enzyme. A pH-rate profile showed that maximum catalysis is achieved above pH 8.0. Surprisingly, at 25 °C, k cat is higher when ADP replaces ATP as substrate for ATPPRT but not for HisG S . The HisG S -catalyzed reaction is limited by the chemical step, as suggested by the enhancement of k cat when Mg 2+ was replaced by Mn 2+ , and by the lack of a pre-steady-state burst of product formation. Conversely, the ATPPRT-catalyzed reaction rate is determined by PRATP diffusion from the active site, as gleaned from a substantial solvent viscosity effect. A burst of product formation could be inferred from pre-steady-state kinetics, but the first turnover was too fast to be directly observed. Lowering the temperature to 5 °C allowed observation of the PRATP formation burst by ATPPRT. At this temperature, the single-turnover rate constant was significantly higher than k cat , providing additional evidence for a step after chemistry limiting catalysis by ATPPRT. This demonstrates allosteric activation by HisZ accelerates the chemical step.

Allosteric activation unveils protein-mass modulation of ATP phosphoribosyltransferase product release

“…This was further probed by pre-steady-state kinetics under multiple-turnover conditions. The Ab HisG S reaction had been shown not to have a burst of product formation 46 , which is reproduced here for unlabelled Ab HisG S , whose traces overlap with those for [ 2 H, 13 C, 15 N] Ab HisG S (Supplementary Fig. 5 ).…”

“…With Ab HisG S , the lack of a burst of product formation suggested no step after chemistry is rate-limiting for the reaction. In agreement, replacement of Mg 2+ by Mn 2+ , which leads to more efficient charge balance at the transition state of the related Psychrobacter arcticus ATPPRT, increased Ab HisG S steady-state catalytic constant ( k cat ), as it does P. arcticus HisG S k cat 2 , 45 , suggesting chemistry is the rate-limiting step for the nonactivated enzyme form 46 . On the other hand, with Ab ATPPRT, a burst of product formation was inferred, although it was too fast at 25 °C to observe directly even with rapid kinetics, suggesting a step after chemistry is rate-limiting.…”

“…On the other hand, with Ab ATPPRT, a burst of product formation was inferred, although it was too fast at 25 °C to observe directly even with rapid kinetics, suggesting a step after chemistry is rate-limiting. This was corroborated by high solvent viscosity effects, which showed PRATP diffusion from the enzyme to be rate-determining for k cat 46 . At 5 °C, the burst phase could finally be observed with Ab ATPPRT; moreover, the single-turnover rate constant ( k STO ), which was much higher than k cat , showed the chemical step is allosterically activated more than 1300-fold in Ab ATPPRT as compared with Ab HisG S 46 .…”

“…This was corroborated by high solvent viscosity effects, which showed PRATP diffusion from the enzyme to be rate-determining for k cat 46 . At 5 °C, the burst phase could finally be observed with Ab ATPPRT; moreover, the single-turnover rate constant ( k STO ), which was much higher than k cat , showed the chemical step is allosterically activated more than 1300-fold in Ab ATPPRT as compared with Ab HisG S 46 .…”

“…For A. baumannii ATPPRT, unique among other reported ATPPRTs due to its reaction proceeding via a rapid equilibrium random mechanism 30 , steady-state and pre-steady-state kinetics studies point to chemistry as the rate-limiting step for nonactivated HisG S (henceforth referred to as Ab HisG S ). Allosteric activation by HisZ ( Ab HisZ) disproportionately enhances the chemical step, making product release rate-limiting for the hetero-octameric holoenzyme (henceforth referred to as Ab ATPPRT) 46 . With Ab HisG S , the lack of a burst of product formation suggested no step after chemistry is rate-limiting for the reaction.…”