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 5-phospho-α-d-ribosyl-1-pyrophosphate to produce N¹-(5-phospho-β-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 Mg²⁺ was replaced by Mn²⁺, 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.

Scheme 1. ATPPRT-Catalyzed Nucleophilic Substitution Reaction

Figure 1

Crystal structure of the unliganded AbATPPRT. (A) Two views of the ribbon diagram of the hetero-octamer. AbHisG_S subunits are shown in pink and teal, whereas AbHisZ subunits are in gray, orange, yellow, and blue. (B) Active-site close-up of the overlay between AbHisG_S and PRPP-ATP-bound P. arcticus HisG_S (6FU2) dimers. Side chains are shown as stick models with carbon atoms in blue for AbATPPRT and in gold for P. arcticus ATPPRT. Residue labels follow the AbATPPRT numbering. No electron density was visible for most of the side-chain atoms of Arg53 and Asn179. PRPP and ATP (from the P. arcticus structure) are shown as stick models with carbon atoms in green. The Mg²⁺ (from the P. arcticus structure) is shown as a sphere.

Figure 2

AbATPPRT pH-rate study. (A) Substrate concentration dependence of AbATPPRT initial rate at different pHs. All data points are shown for two independent measurements at each concentration. Lines are best fit to eq 3. (B) AbATPPRT pH-rate profile of k_cat. Data are mean ± fitting error from two independent measurements. Line is best fit to eq 4.

Figure 3

Pre-steady-state kinetics at 25 °C. (A) Approach to the steady-state formation of PRATP by AbHisG_S. (B) Approach to the steady-state formation of PRATP by AbATPPRT. Dashed lines are linear regressions of the data. Controls lacked PRPP.

Figure 4

Solvent viscosity effects on the AbATPPRT-catalyzed reaction. (A) AbATPPRT apparent rate constants for PRATP formation at saturating substrate concentrations in the presence and absence of either glycerol or PEG-8000. All data points are shown for two independent measurements at each concentration. (B) Solvent viscosity effects on k_cat. Data are mean ± SD for four independent measurements (two at each concentration). Line is best fit to eq 5.

Figure 5

Steady-state kinetics with ADP. (A) Substrate concentration-dependence of AbHisG_S initial rates with either ATP or ADP as substrate. (B) Substrate concentration-dependence of AbATPPRT initial rates with either ATP or ADP as substrate. All data points are shown for two independent measurements at each concentration. Lines are best fit to eq 3.

Figure 6

AbHisG_S steady-state kinetics with Mn²⁺ at 25 °C. All data points are shown for two independent measurements at each concentration. Lines are best fit to eq 3.

Figure 7

Pre-steady-state kinetics at 5 °C. (A) Approach to the steady-state formation of PRATP by AbHisG_S. The dashed line is the best fit to eq 6. (B) Approach to the steady-state formation of PRATP by AbATPPRT. The dashed line is the best fit to eq 7. Controls lacked PRPP.

Figure 8

Single-turnover kinetics of AbATPPRT at 5 °C. Black lines are best fit of the data to eq 8, which yielded the apparent rate constants shown.