Crystal structure, steady-state and pre-steady-state kinetics of Acinetobacter baumannii ATP phosphoribosyltransferase
Date
16/01/2024Metadata
Show full item recordAbstract
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, HisGS, is allosterically activated upon binding of the regulatory subunit, HisZ, to form the hetero-octameric holoenzyme (ATPPRT), leading to a large increase in kcat. Here, we present the crystal structure of ATPPRT, along with kinetic investigations of the rate-limiting steps governing catalysis in the nonactivated (HisGS) 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, kcat is higher when ADP replaces ATP as substrate for ATPPRT but not for HisGS. The HisGS-catalyzed reaction is limited by the chemical step, as suggested by the enhancement of kcat 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 kcat, providing additional evidence for a step after chemistry limiting catalysis by ATPPRT. This demonstrates allosteric activation by HisZ accelerates the chemical step.
Citation
Read , B , Cadzow , A , Alphey , M S , Mitchell , J B O & da Silva , R G 2024 , ' Crystal structure, steady-state and pre-steady-state kinetics of Acinetobacter baumannii ATP phosphoribosyltransferase ' , Biochemistry , vol. 63 , no. 2 , pp. 230-240 . https://doi.org/10.1021/acs.biochem.3c00551
Publication
Biochemistry
Status
Peer reviewed
ISSN
0006-2960Type
Journal article
Description
Funding: This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (grant BB/M010996/1) via an EASTBIO Doctoral Training Partnership studentship to B.J.R.Collections
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