The reaction mechanism and inhibition of ATP-PRTase enzymes.

Abstract

Adenosine triphosphate phosphoribosyltransferase (ATP-PRTase) catalyses the first committed step in histidine biosynthesis. This thesis discusses studies towards a better understanding of the enzyme structure, function and inhibition by both natural and non natural ligands, towards the goal of drug development. ATP-PRTase from Campylobacter jejuni and Mycobacterium tuberculosis, which both carry the long ATP-PRTase enzyme, and from Lactococcus lactis which carries the short form ATP-PRTase enzyme were studied using a range of approaches and techniques. The structural basis of inhibition of the C. jejuni ATP-PRTase was investigated. An altered model of inhibition is proposed, based on two crystal structures: an active ATP-bound structure, and an inactive histidine and adenosine monophosphate (AMP)-bound structure. Intrinsic kinetic isotope effect (KIE) values and transition state modelling was performed for all three ATP-PRTase enzymes. The reaction was studied in reverse, using phosphonoacetic acid as an alternative substrate to pyrophosphate to overcome the suppression of KIEs by commitment to catalysis. Radioactive labels were incorporated by coupled enzymatic syntheses at six positions, with the intrinsic KIEs measured using the internal competition method. A proposed dissociative reaction mechanism was supported by moderate primary 14C isotope effects (1.028 to 1.031) and large α-secondary 3H isotope effects (1.147 to 1.250). The transition state models were located with density functional theory at the B3LYP/6-31*G(d,p) level, and refined the reaction mechanism to a DN*AN‡ type. Synthesis and inhibition studies were also performed, using transition state analogue synthesis, and preliminary work with a fragment based lead discovery technique. Three analogues were developed, with one based on the ring flattening effect predicted for the carbocation intermediate (a cyclopentene based analogue), and two based around the charge change of the cationic species (iminoribitol based analogues). The cyclopentene analogue was found have an IC 50 of 48 ± 4 µM for the M. tuberculosis ATP-PRTase enzyme and a Ki of 282 ± 66 µM for C. jejuni ATP-PRTase. The combined results contribute to the understanding of the reaction mechanisms and inhibition of ATP-PRTase enzymes. The results present the first crystallographic observation of ATP bound to the ATP-PRTase enzyme. KIE and transition state modelling of the reaction of Type IV PRTases was successfully demonstrated. Competitive inhibition was observed for an initial transition state analogue.