title: Chemical transformation is not rate-limiting in the reaction catalyzed by Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase.
abstract: 6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP). Because HPPK is essential for microorganisms but is absent from human and animals, the enzyme is an excellent target for developing antimicrobial agent. Thermodynamic analysis shows that Mg(2+) is important not only for the binding of nucleotides but also for the binding of HMDP. Transient kinetic analysis shows that a step or steps after the chemical transformation are rate-limiting in the reaction catalyzed by HPPK. The pre-steady-state kinetics is composed of a burst phase and a steady-state phase. The rate constant for the burst phase is approximately 50 times larger than that for the steady-state phase. The latter is very similar to the k(cat) value measured by steady-state kinetics. A set of rate constants for the individual steps of the HPPK-catalyzed reaction has been determined by a combination of stopped-flow and quench-flow analyses. These results form a thermodynamic and kinetic framework for dissecting the roles of active site residues in the substrate binding and catalysis by HPPK.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.
authors.forename: Yue
authors.initials: Y
authors.lastname: Li
authors.forename: Yunchen
authors.initials: Y
authors.lastname: Gong
authors.forename: Genbin
authors.initials: G
authors.lastname: Shi
authors.forename: Jaroslaw
authors.initials: J
authors.lastname: Blaszczyk
authors.forename: Xinhua
authors.initials: X
authors.lastname: Ji
authors.forename: Honggao
authors.initials: H
authors.lastname: Yan
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008962:Models, Theoretical; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*
chemical_list: C105176:6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-7,8-dihydropteroate synthase; D009097:Multienzyme Complexes; D000255:Adenosine Triphosphate