The roles of a pair of conserved positively charged residues R82 and R92 at a catalytic loop of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) have been investigated by site-directed mutagenesis and biochemical analysis. In the structure of HPPK in complex with ATP and a 6-hydroxymethyl-7,8-dihydropterin (HP) analogue, the guanidinium group of R82 forms two hydrogen bonds with the alpha-phosphate and that of R92 two hydrogen bonds with the beta-phosphate. In the structure of HPPK in complex with alpha,beta-methyleneadenosine triphosphate (AMPCPP, an ATP analogue) and HP, the guanidinium group of R82 has no direct interaction with AMPCPP and that of R92 forms two hydrogen bonds with the alpha-phosphate. Substitution of R82 with alanine caused a decrease in the rate constant for the chemical step by a factor of approximately 380, but there were no significant changes in the binding energy or binding kinetics of either substrate. Substitution of R92 with alanine caused a decrease in the rate constant for the chemical step by a factor of approximately 3.5 x 10(4). The mutation caused no significant changes in the binding energy or binding kinetics of MgATP. It did not cause a significant change in the binding energy of HP either but caused a decrease in the association rate constant for the binding of HP by a factor of approximately 4.5 and a decrease in the dissociation rate constant by a factor of approximately 10. The overall structures of the ternary complexes of both mutants were very similar to the corresponding structure of wild-type HPPK as described in the companion paper. The results suggest that R82 does not contribute to the binding of either substrate, and R92 is dispensable for the binding of MgATP but plays a role in facilitating the binding of HP. Both R82 and R92 are important for catalysis, and R92 plays a critical role in the transition state stabilization.