Kinetic and structural characterization of a product complex of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Escherichia coli.
HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase) catalyses the transfer of pyrophosphate from ATP to HMDP (6-hydroxymethyl-7,8-dihydropterin), to form AMP and DHPPP (6-hydroxymethyl-7,8-dihydropterin pyrophosphate). This transformation is a key step in the biosynthesis of folic acid, and HPPK is consequently a target for antimicrobial drugs. The substrates are known to bind to HPPK in an ordered manner, with ATP binding first followed by HMDP. In the present study we show by isothermal titration calorimetry that the product, DHPPP, can bind to the HPPK apoenzyme with high affinity (equilibrium dissociation constant, K(d)=0.2 microM), but without the enhancement of pterin fluorescence that occurs on binding of HMDP. The transient kinetics of the enzyme can be monitored by measuring the change in the fluorescence of the pterin ring using stopped-flow methods. The fluorescence exhibits a pronounced biphasic behaviour: it initially rises and then declines back to its original level. This behaviour is in agreement with a two-state kinetic model, with the first phase of fluorescence increase associated with HMDP binding to the enzyme, and the second phase with a slow event that occurs after the reaction has taken place. The HPPK-DHPPP and HPPK-DHPPP-AMP complexes were examined by NMR, and the binding site for DHPPP partially mapped from changes in chemical shifts identified from two dimensional 1H/15N heteronuclear single-quantum coherence spectra. The results demonstrate that DHPPP, in contrast to HMDP, is able to bind to the HPPK apoenzyme and suggest that the pyrophosphate moieties on the ligand play an important role in establishment of a high affinity binding site for the pterin ring.
Garçon
Arnaud
A
Department of Biomolecular Sciences, UMIST, Sackville Street, Manchester M60 1QD, UK.
Bermingham
Alun
A
Lian
Lu-Yun
LY
Derrick
Jeremy P
JP
D001665:Binding Sites; D002151:Calorimetry / Q000379:methods; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D017854:Diphosphotransferases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010756:Phosphoric Acids; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D011622:Pterins / Q000737:chemistry / Q000378:metabolism
C492818:6-hydroxymethyl-7,8-dihydropterin; C492819:6-hydroxymethyl-7,8-dihydropterin pyrophosphate; D011756:Diphosphates; D029968:Escherichia coli Proteins; D010756:Phosphoric Acids; D011622:Pterins; D017854:Diphosphotransferases; C019684:2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase