Mechanistic determination of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK)

The development of widespread, high level resistance to antibiotics has made the quest for the discovery or design of new antibacterial agents a matter of some urgency. Tetrahydrofolate is synthesised de novo in micro-organisms and plants via the folic acid biosynthetic pathway whereas mammals acquire this crucial cofactor in their diets. The individual enzymes involved in this pathway are attractive potential targets for the development of new antibacterial agents. 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) an enzyme of the folic acid biosynthetic pathway catalyses the magnesium-dependent pyrophosphorylation of 6-hydroxymethyl-7,8-dihydropterin, utilising ATP to form 6-hydroxymethyl-7,8-dihydropterin pyrophosphate. This substitution reaction occurs via a nucleophilic displacement reaction at the b-phosphoryl centre of ATP. The stereochemical course of the HPPK catalysed reaction has been investigated by substituting ATP for an analogue that is chiral at the b-phosphoryl centre by virtue of sulphur and 18O substitution. The determination of whether the configuration at this centre is inverted or retained as a result of nucleophilic substitution required the development of a new configurational analysis based on 18O isotopic shifts seen in 31P NMR spectroscopy. This method has allowed us to determine that the configuration at the chiral b-phosphoryl centre has been inverted as a result of pyrophosphoryl transfer. This suggests a mechanism that proceeds via a single in-line associative nucleophilic displacement reaction. The alternative mechanism resulting in retention of configuration at the chiral b-phosphoryl centre would require a double displacement (via two consecutive inversions of configuration) involving a covalent phosphoenzyme intermediate. The observation that HPPK facilitates the pyrophosphorylation of 6-hydroxtmethyl-7,8-dihyropterin via a single in-line associative nucleophilic displacement reaction on the Pb of ATP using the 6-hydroxy group of the pterin as a nucleophile will be of use in the design of inhibitors with a potential therapeutic value.