Equilibrium and kinetic studies of substrate binding to 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase from Escherichia coli

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the py rophosphorylation of 6-hydroxymethyl-7,8-dihydropterin (HMDP) by ATP to for m 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, an intermediate in the p athway for folic acid biosynthesis. The enzyme has been identified as a pot ential target for antimicrobial drugs. Equilibrium binding studies showed t hat Escherichia coli HPPK-bound ATP or the nonhydrolyzable ATP analogue alp ha,beta-methyleneadenosine triphosphate (AMPCPP) with high affinity. The fl uorescent ATP analogue 2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphos phate (MANT-ATP) exhibited a substantial fluorescence enhancement upon bind ing to HPPK with an equilibrium dissociation constant comparable with that for ATP (10.4 and 4.5 mu M, respectively). The apoenzyme did not bind the s econd substrate HMDP, however, unless AMPCPP was present, suggesting that t he enzyme binds ATP first, followed by HMDP. Equilibrium titration of HPPK into HMDP and AMPCPP showed an enhancement of fluorescence from the pterin ring of the substrate, and a dissociation constant of 36 nM was deduced for HMDP binding to the HPPK.AMPCPP binary complex, Stopped flow fluorimetry m easurements showed that the rate constants for the binding of MANT-ATP and AMPCPP to HPPK were relatively slow (3.9 x 10(5) and 1.05 x 10(5) M-1 s(-1) , respectively) compared with the on rate for binding of HMDP to the HPPK.A MPCPP binary complex. The significance of these results with respect to the crystal structures of HPPK is discussed.